Methods for achieving therapeutically effective doses of anti-CD47 agents

ABSTRACT

Methods are provided for treating a subject with a therapeutic dose of anti-CD47 agent by administering a primer agent prior to administering a therapeutically effective dose of an anti-CD47 agent to the subject.

CROSS REFERENCE

This application claims benefit and is a Continuation of applicationSer. No. 14/769,069 filed Aug. 19, 2015, which is a 371 application andclaims the benefit of PCT Application No. PCT/US2014/018743, filed Feb.26, 2014, which claims benefit of U.S. Provisional Patent ApplicationNo. 61/800,102, filed Mar. 15, 2013, which applications are incorporatedherein by reference in their entirety.

BACKGROUND

Turnover of cells begins with the induction of an apoptotic program orother cellular changes that mark them for removal, and the subsequentrecognition of markers by phagocytes, including macrophages, dendriticcells, and the like. This process requires a specific and selectiveremoval of unwanted cells. Unlike healthy cells, the unwanted/aged/dyingcells display markers or ligands called “eat-me” signals, i.e. “alteredself”, which can in turn be recognized by receptors on the phagocytes.Healthy cells may display “don't eat-me” signals that actively inhibitphagocytosis; these signals are either downregulated in the dying cells,are present in an altered conformation or they are superseded by theupregulation of “eat-me” or pro-phagocytic signals. The cell surfaceprotein CD47 on healthy cells and its engagement of a phagocytereceptor, SIRPα, constitutes a key “don't eat-me” signal that can turnoff engulfment mediated by multiple modalities, including apoptotic cellclearance and FcR mediated phagocytosis. Blocking the CD47 mediatedengagement of SIRPα on a phagocyte, or the loss of CD47 expression inknockout mice, can cause removal of live cells and non-agederythrocytes. Blocking SIRPα also allows engulfment of targets that arenot normally phagocytosed, for those cells where pre-phagocytic signalsare also present.

CD47 is a broadly expressed transmembrane glycoprotein with a singleIg-like domain and five membrane spanning regions, which functions as acellular ligand for SIRPα with binding mediated through the NH2-terminalV-like domain of SIRPα. SIRPα is expressed primarily on myeloid cells,including macrophages, granulocytes, myeloid dendritic cells (DCs), mastcells, and their precursors, including hematopoietic stem cells.Structural determinants on SIRPα that mediate CD47 binding are discussedby Lee et al. (2007) J. Immunol. 179:7741-7750; Hatherley et al. (2007)J.B.C. 282:14567-75; and the role of SIRPα cis dimerization in CD47binding is discussed by Lee et al. (2010) J.B.C. 285:37953-63. Inkeeping with the role of CD47 to inhibit phagocytosis of normal cells,there is evidence that it is transiently upregulated on hematopoieticstem cells (HSCs) and progenitors just prior to and during theirmigratory phase, and that the level of CD47 on these cells determinesthe probability that they are engulfed in vivo.

Programmed cell death (PCD) and phagocytic cell removal are common waysthat an organism responds in order to remove damaged, precancerous, orinfected cells. Thus, the cells that survive this organismal response(e.g., cancerous cells, chronically infected cells, etc.) have devisedways to evade PCD and phagocytic cell removal. CD47, the “don't eat me”signal, is constitutively upregulated on a wide variety of diseasedcells, cancer cells, and infected cells, allowing these cells to evadephagocytosis. Anti-CD47 agents that block the interaction between CD47on one cell (e.g., a cancer cell, an infected cell, etc.) and SIRPα onanother cell (e.g., a phagocytic cell) counteract the increase of CD47expression and facilitate the phagocytosis of the cancer cell and/or theinfected cell. Thus, anti-CD47 agents can be used to treat and/orprotect against a wide variety of conditions/disorders.

However, an initial high dose of an anti-CD47 agent can cause adose-dependent loss of red blood cells (RBCs) in mice and non-humanprimate (NHP) models. The severity of this anemia can preclude the useof higher doses that are required to achieve sustained serumconcentrations associated with therapeutic efficacy. The presentinvention provides methods by which the erythrocyte toxicity ofanti-CD47 agents is mitigated, thereby enabling treatment withtherapeutically effective amounts of anti-CD47 agents.

SUMMARY OF THE INVENTION

Methods are provided for treating an individual with a therapeutic doseof anti-CD47 agent by administering a primer agent prior toadministering a therapeutically effective dose of an anti-CD47 agent tothe individual. In some embodiments, the methods of the invention finduse in optimizing therapies aimed at modulating CD47-mediatedphagocytosis. In some such embodiments, the individual is being treatedwith a dose of anti-CD47 agent for cancer. In other embodiments theindividual is being treated with a dose of anti-CD47 agent for infectionwith an intracellular pathogen. In the subject methods, atherapeutically effective dose of an anti-CD47 agent is administeredfrom about 3 days to about 21 days after administering a primer agent.

In some embodiments of the invention, two or more primer agents areadministered. Suitable primer agents include anerythropoiesis-stimulating agent (ESA), and/or a priming dose of ananti-CD47 agent.

An anti-CD47 agent for use in the methods of the invention interfereswith binding between CD47 present on a target cell, including withoutlimitation a cancer cell, a cell infected with an intracellularpathogen, a stem cell, etc., to SIRPα present on a phagocytic cell.Generally both such cells are present in the individual being treated.Such methods, in the presence of a pro-phagocytic signal, can increasephagocytosis of the target cell. The subject methods can be used totreat a subject for any disease susceptible to blockade of CD47-mediatedSIRPα signaling. Suitable anti-CD47 agents include soluble SIRPαpolypeptides; soluble CD47; anti-CD47 antibodies, anti-SIRPα antibodies,and the like, where the term antibodies encompasses antibody fragmentsand variants thereof, as known in the art.

Therapeutic doses of anti-CD47 agents as described above can lead to aloss of erythrocytes (RBCs) and anemia. The methods of the inventionaddress this problem, and surprisingly show that a primer agent, as usedherein, significantly reduces toxicity due to loss of erythrocytes.Without being bound by theory, it is believed that the primer agentincreases production of reticulocytes (immature RBC), which may be moreresistant to CD47 mediated phagocytosis and therefore are lesssusceptible to loss during subsequent administration of the anti-CD47agent.

Certain embodiments of the invention optionally include a step ofdetermining responsiveness of an individual to administration of theprimer agent. For example, a reticulocyte count, or a decrease inhemoglobin, can be used to determine whether administration of theprimer agent increased production of reticulocytes. A reticulocyte countcan be performed prior to and following primer agent administration,allowing for a period of time between counts that is effective for anincrease in reticulocytes. Alternatively, any suitable methods fordetermination of increased erythropoiesis can be used.

Following administration of the priming agent, and allowing a period oftime effective for an increase in reticulocyte production, a therapeuticdose of an anti-CD47 agent is administered. The therapeutic dose can beadministered in number of different ways. In some embodiments, two ormore therapeutically effective doses are administered after a primeragent is administered. In some embodiments a therapeutically effectivedose of an anti-CD47 agent is administered as two or more doses ofescalating concentration, in others the doses are equivalent.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is best understood from the following detailed descriptionwhen read in conjunction with the accompanying drawings. The patent orapplication file contains at least one drawing executed in color. Copiesof this patent or patent application publication with color drawing(s)will be provided by the Office upon request and payment of the necessaryfee. It is emphasized that, according to common practice, the variousfeatures of the drawings are not to-scale. On the contrary, thedimensions of the various features are arbitrarily expanded or reducedfor clarity. Included in the drawings are the following figures.

FIG. 1A-1B presents percent change in hematocrit (HCT) and percentchange hemoglobin after a single 250 μg IP injection of MIAP410 (IgG1isotype) or MIAP470 (IgG2a isotype) was administered to wild type mice.

FIG. 2 presents percent change in hemoglobin after a single 250 μg IPinjection of MIAP410 (IgG1 isotype) or MIAP470 (IgG2a isotype) wasadministered to CD47^(−/−) mice.

FIG. 3A-3B presents percent change in hematocrit (HCT) and percentchange hemoglobin after IP injections of 250 μg of control mouse IgG,MIAP410, or MIAP740 were administered to wild type mice every 3 days.

FIG. 4 depicts a sequence alignment between human and macaque CD47 inthe Ig-like extracellular domain. Human CD47ECD (SEQ ID NO: 4); Cyno(macaque) CD47ECD (SEQ ID NO: 5).

FIG. 5 demonstrates that Hu5F9-G4 recognizes human and cynomolgus CD47,but not mouse CD47. ELISA was performed by coating an anti-mouse Fcspecific antibody, followed by adding human, mouse, and cyno CD47-mFcfusion proteins. An irrelevant mouse Fc (mFc) fusion protein was used asa negative control. Hu5F9-G4 was then added. Bound antibody was detectedusing HRP-conjugated anti-human Kappa antibody.

FIG. 6A-6B presents a summary of binding constants measured for Hu5F9-G4binding. SPR binding studies were performed on a BioRad ProteOn XPR36system using a GLM sensor chip. Binding data were collected at 25° C.Response data were globally fit using a 1:1 interaction model. Thenumber in parentheses represents the standard error in the last reporteddigit. (FIG. 6A) Binding to human CD47. (FIG. 6B) Binding to cynomolgusCD47.

FIG. 7A-7B presents data from Non-Human Primate Hu5F9-G4 toxicokineticstudies.

Cynomolgus NHP were administered Hu5F9-G4 by single doses at theindicated levels. (FIG. 7A) Anemia developed in a dose-dependent manner,but resolved spontaneously. The shaded bar indicates the range ofhemoglobin that indicates the need for transfusion in humans. (FIG. 7B)Pharmacokinetic (PK) analysis by measurement of serum levels indicated ashort-half life with therapeutic levels achieved by 10 and 30 mg/kg, butnot the other doses.'

FIG. 8A-8B presents data from a Non-Human Primate Hu5F9-G4 doseescalation toxicokinetic study. Cynomolgus NHP that received either nopre-treatment or pre-treatment with a single dose of EPO wereadministered Hu5F9-G4 in a dose escalation study with the doses and timepoints indicated. (FIG. 8A) Hemoglobin was serially measured to monitoranemia. (FIG. 8B) Serum was screened for the level of Hu5F9-G4 by ELISAto determine pharmacokinetics (PK). In panel (FIG. 8A), the shaded barindicates the range of hemoglobin in humans that tends to triggertransfusion. In panel (FIG. 8B), the shaded bar indicates the range ofserum Hu5F9-G4 associated with potent efficacy in xenograft studies.

FIG. 9A-9B presents data from a Non-Human Primate Hu5F9-G4Loading-Maintenance Dose Toxicokinetic Study. Cynomolgus NHP received aloading dose (LD) (i.e., a priming dose) on day 1 of either 1 mg/kg or 3mg/kg and then maintenance doses (MD) of either 10 or 30 mg/kg at theindicated time points. 2 NHPs (solid line and dashed line) were used ineach experimental group. (FIG. 9A) Hemoglobin was serially measured tomonitor anemia. (FIG. 9B) Serum was screened for the level of Hu5F9-G4by ELISA to determine pharmacokinetics. In panel (FIG. 9A), the shadedbar indicates the range of hemoglobin in humans that might triggertransfusion. In panel (FIG. 9B), the shaded bar indicates the range ofserum Hu5F9-G4 associated with potent efficacy against primary human AMLin xenograft studies (i.e., the range of therapeutically effective serumlevels).

FIG. 10 presents reticulocyte count data demonstrating the level ofreticulocytosis associated with various doses of an anti-CD47 agent(hu5F9-G4 antibody in this case).

FIG. 11A-11B demonstrates that Hu5F9-G4 inhibits tumor growth andmetastasis. (FIG. 11A) Hu5F9-G4 fully eliminates bladder cancer inxenotransplantation assays. (FIG. 11B) Hu5F9-G4 prevents human prostatecancer metastasis in vivo.

FIG. 12A-12D demonstrates that Hu5F9-G4 eliminates establishedmetastases. (FIG. 12A-12B) Hu5F9-G4 eliminates metastatic breast cancercells in the lungs (FIG. 12A) and brain (FIG. 12B). (FIG. 12C) Hu5F9-G4inhibits regrowth of resected breast tumors. (FIG. 12D) Serumconcentrations of hu5F9-G4 associated with therapeutic efficacy. Thus,humanized antibodies (e.g., hu5F9-G4), have the same general propertiesrelated to the treatment of disease (e.g., cancer or chronic infection)as the non-humanized antibodies and the subject methods will beeffective when using a humanized antibody (e.g., anti-CD47 antibody) totreat cancer and/or to treat chronic infection.

FIG. 13 depicts the study design described in Example 4.

FIG. 14 depicts hemoglobin level data for all cohorts over the durationof the study described in Example 4 (also see FIG. 13).

FIG. 15 depicts the pharmacokinetic profile of Hu5F9-G4 (humanizedanti-CD47 antibody) in all cohorts of the study described in Example 4(also see FIG. 13).

DETAILED DESCRIPTION

The present invention relates to methods of treating a subject with atherapeutic dose of anti-CD47 agent by first administering a primeragent.

Before the present methods and compositions are described, it is to beunderstood that this invention is not limited to particular method orcomposition described, as such may, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting, since the scope of the present invention will be limited onlyby the appended claims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimits of that range is also specifically disclosed. Each smaller rangebetween any stated value or intervening value in a stated range and anyother stated or intervening value in that stated range is encompassedwithin the invention. The upper and lower limits of these smaller rangesmay independently be included or excluded in the range, and each rangewhere either, neither or both limits are included in the smaller rangesis also encompassed within the invention, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either or both of those includedlimits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, some potential andpreferred methods and materials are now described. All publicationsmentioned herein are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. It is understood that the present disclosuresupersedes any disclosure of an incorporated publication to the extentthere is a contradiction.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentinvention. Any recited method can be carried out in the order of eventsrecited or in any other order which is logically possible.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “acell” includes a plurality of such cells and reference to “the peptide”includes reference to one or more peptides and equivalents thereof, e.g.polypeptides, known to those skilled in the art, and so forth.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

Definitions

Anti-CD47 agent. As used herein, the term “anti-CD47 agent” refers toany agent that reduces the binding of CD47 (e.g., on a target cell) toSIRPα (e.g., on a phagocytic cell). Non-limiting examples of suitableanti-CD47 reagents include SIRPα reagents, including without limitationhigh affinity SIRPα polypeptides, anti-SIRPα antibodies, soluble CD47polypeptides, and anti-CD47 antibodies or antibody fragments. In someembodiments, a suitable anti-CD47 agent (e.g. an anti-CD47 antibody, aSIRPα reagent, etc.) specifically binds CD47 to reduce the binding ofCD47 to SIRPα. In some embodiments, a suitable anti-CD47 agent (e.g., ananti-SIRPα antibody, a soluble CD47 polypeptide, etc.) specificallybinds SIRPα to reduce the binding of CD47 to SIRPα. A suitable anti-CD47agent that binds SIRPα does not activate SIRPα (e.g., in theSIRPα-expressing phagocytic cell). The efficacy of a suitable anti-CD47agent can be assessed by assaying the agent (further described below).In an exemplary assay, target cells are incubated in the presence orabsence of the candidate agent. An agent for use in the methods of theinvention will up-regulate phagocytosis by at least 10% (e.g., at least20%, at least 30%, at least 40%, at least 50%, at least 60%, at least70%, at least 80%, at least 90%, at least 100%, at least 120%, at least140%, at least 160%, at least 180%, or at least 200%) compared tophagocytosis in the absence of the agent. Similarly, an in vitro assayfor levels of tyrosine phosphorylation of SIRPα will show a decrease inphosphorylation by at least 5% (e.g., at least 10%, at least 15%, atleast 20%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, or 100%) compared tophosphorylation observed in absence of the candidate agent.

In some embodiments, the anti-CD47 agent does not activate CD47 uponbinding. When CD47 is activated, a process akin to apoptosis (i.e.,programmed cell death) may occur (Manna and Frazier, Cancer Research,64, 1026-1036, Feb. 1 2004). Thus, in some embodiments, the anti-CD47agent does not directly induce cell death of a CD47-expressing cell.

Some pathogens (e.g., pox viruses, Myxoma virus, Deerpox virus, swinepoxvirus, goatpox virus, sheeppox virus, etc.) express a CD47-analog (i.e.,a CD47 mimic) (e.g., the M128L protein) that acts as a virulence factorto enable infection (Cameron et al., Virology. 2005 Jun. 20;337(1):55-67), and some pathogens induce the expression of endogenousCD47 in the host cell. Cells infected with a pathogen that expresses aCD47-analog may therefore express the pathogen-provided CD47 analogeither exclusively or in combination with endogenous CD47. Thismechanism allows the pathogen to increase CD47 expression (viaexpression of the CD47 analog) in the infected cell with or withoutincreasing the level of endogenous CD47. In some embodiments, ananti-CD47 agent (e.g., anti-CD47 antibody, a SIRPα reagent, a SIRPαantibody, a soluble CD47 polypeptide, etc.) can reduce the binding of aCD47 analog (i.e., a CD47 mimic) to SIRPα. In some cases, a suitableanti-CD47 agent (e.g., a SIRPα reagent, an anti-CD47 antibody, etc.) canbind a CD47 analog (i.e., a CD47 mimic) to reduce the binding of theCD47 analog to SIRPα. In some cases, a suitable anti-CD47 agent (e.g.,an anti-SIRPα antibody, a soluble CD47 polypeptide, etc.) can bind toSIRPα. A suitable anti-CD47 agent that binds SIRPα does not activateSIRPα (e.g., in the SIRPα-expressing phagocytic cell). An anti-CD47agent can be used in any of the methods provided herein when thepathogen is a pathogen that provides a CD47 analog. In other words theterm “CD47,” as used herein, encompasses CD47 as well as CD47 analogs(i.e., CD47 mimics).

SIRPα reagent. A SIRPα reagent comprises the portion of SIRPα that issufficient to bind CD47 at a recognizable affinity, which normally liesbetween the signal sequence and the transmembrane domain, or a fragmentthereof that retains the binding activity. A suitable SIRPα reagentreduces (e.g., blocks, prevents, etc.) the interaction between thenative proteins SIRPα and CD47. The SIRPα reagent will usually compriseat least the dl domain of SIRPα. In some embodiments, a SIRPα reagent isa fusion protein, e.g., fused in frame with a second polypeptide. Insome embodiments, the second polypeptide is capable of increasing thesize of the fusion protein, e.g., so that the fusion protein will not becleared from the circulation rapidly. In some embodiments, the secondpolypeptide is part or whole of an immunoglobulin Fc region. The Fcregion aids in phagocytosis by providing an “eat me” signal, whichenhances the block of the “don't eat me” signal provided by the highaffinity SIRPα reagent. In other embodiments, the second polypeptide isany suitable polypeptide that is substantially similar to Fc, e.g.,providing increased size, multimerization domains, and/or additionalbinding or interaction with Ig molecules.

In some embodiments, a subject anti-CD47 agent is a “high affinity SIRPαreagent”, which includes SIRPα -derived polypeptides and analogsthereof. High affinity SIRPα reagents are described in internationalapplication PCT/US13/21937, which is hereby specifically incorporated byreference. High affinity SIRPα reagents are variants of the native SIRPαprotein. In some embodiments, a high affinity SIRPα reagent is soluble,where the polypeptide lacks the SIRPα transmembrane domain and comprisesat least one amino acid change relative to the wild-type SIRPα sequence,and wherein the amino acid change increases the affinity of the SIRPαpolypeptide binding to CD47, for example by decreasing the off-rate byat least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold,at least 500-fold, or more.

A high affinity SIRPα reagent comprises the portion of SIRPα that issufficient to bind CD47 at a recognizable affinity, e.g., high affinity,which normally lies between the signal sequence and the transmembranedomain, or a fragment thereof that retains the binding activity. Thehigh affinity SIRPα reagent will usually comprise at least the dl domainof SIRPα with modified amino acid residues to increase affinity. In someembodiments, a SIRPα variant of the present invention is a fusionprotein, e.g., fused in frame with a second polypeptide. In someembodiments, the second polypeptide is capable of increasing the size ofthe fusion protein, e.g., so that the fusion protein will not be clearedfrom the circulation rapidly. In some embodiments, the secondpolypeptide is part or whole of an immunoglobulin Fc region. The Fcregion aids in phagocytosis by providing an “eat me” signal, whichenhances the block of the “don't eat me” signal provided by the highaffinity SIRPα reagent. In other embodiments, the second polypeptide isany suitable polypeptide that is substantially similar to Fc, e.g.,providing increased size, multimerization domains, and/or additionalbinding or interaction with Ig molecules. The amino acid changes thatprovide for increased affinity are localized in the dl domain, and thushigh affinity SIRPα reagents comprise a dl domain of human SIRPα, withat least one amino acid change relative to the wild-type sequence withinthe dl domain. Such a high affinity SIRPα reagent optionally comprisesadditional amino acid sequences, for example antibody Fc sequences;portions of the wild-type human SIRPα protein other than the dl domain,including without limitation residues 150 to 374 of the native proteinor fragments thereof, usually fragments contiguous with the dl domain;and the like. High affinity SIRPα reagents may be monomeric ormultimeric, i.e. dimer, trimer, tetramer, etc.

Anti-CD47 antibodies. In some embodiments, a subject anti-CD47 agent isan antibody that specifically binds CD47 (i.e., an anti-CD47 antibody)and reduces the interaction between CD47 on one cell (e.g., an infectedcell) and SIRPα on another cell (e.g., a phagocytic cell). In someembodiments, a suitable anti-CD47 antibody does not activate CD47 uponbinding. Non-limiting examples of suitable antibodies include clonesB6H12, 5F9, 8B6, and C3 (for example as described in InternationalPatent Publication WO 2011/143624, herein specifically incorporated byreference). Suitable anti-CD47 antibodies include fully human, humanizedor chimeric versions of such antibodies. Humanized antibodies (e.g.,hu5F9-G4) are especially useful for in vivo applications in humans dueto their low antigenicity. Similarly caninized, felinized, etc.antibodies are especially useful for applications in dogs, cats, andother species respectively. Antibodies of interest include humanizedantibodies, or caninized, felinized, equinized, bovinized, porcinized,etc., antibodies, and variants thereof.

Anti-SIRPα antibodies. In some embodiments, a subject anti-CD47 agent isan antibody that specifically binds SIRPα (i.e., an anti-SIRPα antibody)and reduces the interaction between CD47 on one cell (e.g., an infectedcell) and SIRPα on another cell (e.g., a phagocytic cell). Suitableanti-SIRPα antibodies can bind SIRPα without activating or stimulatingsignaling through SIRPα because activation of SIRPα would inhibitphagocytosis. Instead, suitable anti-SIRPα antibodies facilitate thepreferential phagocytosis of inflicted cells over normal cells. Thosecells that express higher levels of CD47 (e.g., infected cells) relativeto other cells (non-infected cells) will be preferentially phagocytosed.Thus, a suitable anti-SIRPα antibody specifically binds SIRPα (withoutactivating/stimulating enough of a signaling response to inhibitphagocytosis) and blocks an interaction between SIRPα and CD47. Suitableanti-SIRPα antibodies include fully human, humanized or chimericversions of such antibodies. Humanized antibodies are especially usefulfor in vivo applications in humans due to their low antigenicity.Similarly caninized, felinized, etc. antibodies are especially usefulfor applications in dogs, cats, and other species respectively.Antibodies of interest include humanized antibodies, or caninized,felinized, equinized, bovinized, porcinized, etc., antibodies, andvariants thereof.

Soluble CD47 polypeptides. In some embodiments, a subject anti-CD47agent is a soluble CD47 polypeptide that specifically binds SIRPα andreduces the interaction between CD47 on one cell (e.g., an infectedcell) and SIRPα on another cell (e.g., a phagocytic cell). A suitablesoluble CD47 polypeptide can bind SIRPα without activating orstimulating signaling through SIRPα because activation of SIRPα wouldinhibit phagocytosis. Instead, suitable soluble CD47 polypeptidesfacilitate the preferential phagocytosis of infected cells overnon-infected cells. Those cells that express higher levels of CD47(e.g., infected cells) relative to normal, non-target cells (normalcells) will be preferentially phagocytosed. Thus, a suitable solubleCD47 polypeptide specifically binds SIRPα without activating/stimulatingenough of a signaling response to inhibit phagocytosis.

In some cases, a suitable soluble CD47 polypeptide can be a fusionprotein (for example as structurally described in US Patent PublicationUS20100239579, herein specifically incorporated by reference). However,only fusion proteins that do not activate/stimulate SIRPα are suitablefor the methods provided herein. Suitable soluble CD47 polypeptides alsoinclude any peptide or peptide fragment comprising variant or naturallyexisting CD47 sequences (e.g., extracellular domain sequences orextracellular domain variants) that can specifically bind SIRPα andinhibit the interaction between CD47 and SIRPα without stimulatingenough SIRPα activity to inhibit phagocytosis.

In certain embodiments, soluble CD47 polypeptide comprises theextracellular domain of CD47, including the signal peptide (SEQ IDNO:2), such that the extracellular portion of CD47 is typically 142amino acids in length, and has the amino acid sequence set forth in SEQID NO:3. The soluble CD47 polypeptides described herein also includeCD47 extracellular domain variants that comprise an amino acid sequenceat least 65%-75%, 75%-80%, 80-85%, 85%-90%, or 95%-99% (or any percentidentity not specifically enumerated between 65% to 100%), whichvariants retain the capability to bind to SIRPα without stimulatingSIRPα signaling.

In certain embodiments, the signal peptide amino acid sequence may besubstituted with a signal peptide amino acid sequence that is derivedfrom another polypeptide (e.g., for example, an immunoglobulin orCTLA4). For example, unlike full-length CD47, which is a cell surfacepolypeptide that traverses the outer cell membrane, the soluble CD47polypeptides are secreted; accordingly, a polynucleotide encoding asoluble CD47 polypeptide may include a nucleotide sequence encoding asignal peptide that is associated with a polypeptide that is normallysecreted from a cell.

In other embodiments, the soluble CD47 polypeptide comprises anextracellular domain of CD47 that lacks the signal peptide. In anexemplary embodiment, the CD47 extracellular domain lacking the signalpeptide has the amino acid sequence set forth in SEQ ID NO:1 (124 aminoacids). As described herein, signal peptides are not exposed on the cellsurface of a secreted or transmembrane protein because either the signalpeptide is cleaved during translocation of the protein or the signalpeptide remains anchored in the outer cell membrane (such a peptide isalso called a signal anchor). The signal peptide sequence of CD47 isbelieved to be cleaved from the precursor CD47 polypeptide in vivo.

In other embodiments, a soluble CD47 polypeptide comprises a CD47extracellular domain variant. Such a soluble CD47 polypeptide retainsthe capability to bind to SIRPα without stimulating SIRPα signaling. TheCD47 extracellular domain variant may have an amino acid sequence thatis at least 65%-75%, 75%-80%, 80-85%, 85%-90%, or 95%-99% identical(which includes any percent identity between any one of the describedranges) to SEQ ID NO:1.

The terms “treatment”, “treating”, “treat” and the like are used hereinto generally refer to obtaining a desired pharmacologic and/orphysiologic effect. The effect can be prophylactic in terms ofcompletely or partially preventing a disease or symptom(s) thereofand/or may be therapeutic in terms of a partial or completestabilization or cure for a disease and/or adverse effect attributableto the disease. The term “treatment” encompasses any treatment of adisease in a mammal, particularly a human, and includes: (a) preventingthe disease and/or symptom(s) from occurring in a subject who may bepredisposed to the disease or symptom but has not yet been diagnosed ashaving it; (b) inhibiting the disease and/or symptom(s), i.e., arrestingtheir development; or (c) relieving the disease symptom(s), i.e.,causing regression of the disease and/or symptom(s). Those in need oftreatment include those already inflicted (e.g., those with cancer,those with an infection, etc.) as well as those in which prevention isdesired (e.g., those with increased susceptibility to cancer, those withan increased likelihood of infection, those suspected of having cancer,those suspected of harboring an infection, etc.).

A target cell may be a cell that is “inflicted”, where the term“inflicted” is used herein to refer to a subject with symptoms, anillness, or a disease that can be treated with an anti-CD47 agent. An“inflicted” subject can have cancer, can harbor an infection (e.g., achronic infection), and other hyper-proliferative conditions, forexample sclerosis, fibrosis, and the like, etc. “Inflicted cells” may bethose cells that cause the symptoms, illness, or disease. Asnon-limiting examples, the inflicted cells of an inflicted patient canbe cancer cells, infected cells, and the like. One indication that anillness or disease can be treated with an anti-CD47 agent is that theinvolved cells (i.e., the inflicted cells, e.g., the cancerous cells,the infected cells, etc.) express an increased level of CD47 compared tonormal cells of the same cell type.

A therapeutic treatment is one in which the subject is inflicted priorto administration and a prophylactic treatment is one in which thesubject is not inflicted prior to administration. In some embodiments,the subject has an increased likelihood of becoming inflicted or issuspected of being inflicted prior to treatment. In some embodiments,the subject is suspected of having an increased likelihood of becominginflicted.

Examples of symptoms, illnesses, and/or diseases that can be treatedwith an anti-CD47 agent include, but are not limited to cancer andinfection (e.g., chronic infection). As used herein “cancer” includesany form of cancer (e.g., leukemia; acute myeloid leukemia (AML); acutelymphoblastic leukemia (ALL); metastasis; minimal residual disease;solid tumor cancers, e.g., breast, bladder, colon, ovarian,glioblastoma, leiomyosarcoma, and head & neck squamous cell carcinomas;etc.). Any cancer, where the cancer cells exhibit increased expressionof CD47 compared to non-cancer cells, is a suitable cancer to be treatedby the subject methods and compositions.

As used herein, the term “infection” refers to any state in at least onecell of an organism (i.e., a subject) is infected by an infectious agent(e.g., a subject has an intracellular pathogen infection, e.g., achronic intracellular pathogen infection). As used herein, the term“infectious agent” refers to a foreign biological entity (i.e. apathogen) that induces increased CD47 expression in at least one cell ofthe infected organism. For example, infectious agents include, but arenot limited to bacteria, viruses, protozoans, and fungi. Intracellularpathogens are of particular interest. Infectious diseases are disorderscaused by infectious agents. Some infectious agents cause norecognizable symptoms or disease under certain conditions, but have thepotential to cause symptoms or disease under changed conditions. Thesubject methods can be used in the treatment of chronic pathogeninfections, for example including but not limited to viral infections,e.g. retrovirus, lentivirus, hepadna virus, herpes viruses, pox viruses,human papilloma viruses, etc.; intracellular bacterial infections, e.g.Mycobacterium, Chlamydophila, Ehrlichia, Rickettsia, Brucella,Legionella, Francisella, Listeria, Coxiella, Neisseria, Salmonella,Yersinia sp, Helicobacter pylori etc.; and intracellular protozoanpathogens, e.g. Plasmodium sp, Trypanosoma sp., Giardia sp., Toxoplasmasp., Leishmania sp., etc.

As used herein, a “target cell” is a cell expressing CD47 on thesurface, where masking or otherwise altering the CD47 positive phenotype(e.g., by administration of an anti-CD47 agent) results in increasedphagocytosis. Usually a target cell is a mammalian cell, for example ahuman cell.

The terms “recipient”, “individual”, “subject”, “host”, and “patient”,are used interchangeably herein and refer to any mammalian subject forwhom diagnosis, treatment, or therapy is desired, particularly humans.“Mammal” for purposes of treatment refers to any animal classified as amammal, including humans, domestic and farm animals, and zoo, sports, orpet animals, such as dogs, horses, cats, cows, sheep, goats, pigs, etc.Preferably, the mammal is human.

A “therapeutically effective dose” or “therapeutic dose” is an amountsufficient to effect desired clinical results (i.e., achieve therapeuticefficacy). A therapeutically effective dose can be administered in oneor more administrations. For purposes of this invention, atherapeutically effective dose of an anti-CD47 agent is an amount thatis sufficient to palliate, ameliorate, stabilize, reverse, prevent, slowor delay the progression of the disease state (e.g., cancer or chronicinfection) by increasing phagocytosis of a target cell (e.g., a targetcell). Thus, a therapeutically effective dose of an anti-CD47 agentreduces the binding of CD47 on an target cell, to SIRPα on a phagocyticcell, at an effective dose for increasing the phagocytosis of the targetcell.

In some embodiments, a therapeutically effective dose leads to sustainedserum levels of anti-CD47 agent (e.g., an anti-CD47 antibody) of about40 μg/ml or more (e.g, about 50 ug/ml or more, about 60 ug/ml or more,about 75 ug/ml or more, about 100 ug/ml or more, about 125 ug/ml ormore, or about 150 ug/ml or more). In some embodiments, atherapeutically effective dose leads to sustained serum levels ofanti-CD47 agent (e.g., an anti-CD47 antibody) that range from about 40μg/ml to about 300 ug/ml (e.g, from about 40 ug/ml to about 250 ug/ml,from about 40 ug/ml to about 200 ug/ml, from about 40 ug/ml to about 150ug/ml, from about 40 ug/ml to about 100 ug/ml, from about 50 ug/ml toabout 300 ug/ml, from about 50 ug/ml to about 250 ug/ml, from about 50ug/ml to about 200 ug/ml, from about 50 ug/ml to about 150 ug/ml, fromabout 75 ug/ml to about 300 ug/ml from about 75 ug/ml to about 250ug/ml, from about 75 ug/ml to about 200 ug/ml, from about 75 ug/ml toabout 150 ug/ml, from about 100 ug/ml to about 300 ug/ml, from about 100ug/ml to about 250 ug/ml, or from about 100 ug/ml to about 200 ug/ml).In some embodiments, a therapeutically effective dose for treating solidtumors leads to sustained serum levels of anti-CD47 agent (e.g., ananti-CD47 antibody) of about 100 μg/ml or more (e.g., sustained serumlevels that range from about 100 ug/ml to about 200 ug/ml). In someembodiments, a therapeutically effective dose for treating non-solidtumors (e.g., acute myeloid leukemia (AML)) leads to sustained serumlevels of anti-CD47 agent (e.g., an anti-CD47 antibody) of about 50μg/ml or more (e.g., sustained serum levels of 75 μg/ml or more; orsustained serum levels that range from about 50 ug/ml to about 150ug/ml).

Accordingly, a single therapeutically effective dose or a series oftherapeutically effective doses would be able to achieve and maintain aserum level of anti-CD47 agent. A therapeutically effective dose of ananti-CD47 agent can depend on the specific agent used, but is usuallyabout 8 mg/kg body weight or more (e.g., about 8 mg/kg or more, about 10mg/kg or more, about 15 mg/kg or more, about 20 mg/kg or more, about 25mg/kg or more, about 30 mg/kg or more, about 35 mg/kg or more, or about40 mg/kg or more), or from about 10 mg/kg to about 40 mg/kg (e.g., fromabout 10 mg/kg to about 35 mg/kg, or from about 10 mg/kg to about 30mg/kg). The dose required to achieve and/or maintain a particular serumlevel is proportional to the amount of time between doses and inverselyproportional to the number of doses administered. Thus, as the frequencyof dosing increases, the required dose decreases. The optimization ofdosing strategies will be readily understood and practiced by one ofordinary skill in the art.

A sub-therapeutic dose is a dose (i.e., an amount) that is notsufficient to effect the desired clinical results. For example, asub-therapeutic dose of an anti-CD47 agent is an amount that is notsufficient to palliate, ameliorate, stabilize, reverse, prevent, slow ordelay the progression of the disease state (e.g., cancer, infection,inflammation, etc.). In some cases, it is desirable to use asub-therapeutic dose of an anti-CD47 agent as a primer agent (describedin more detail below). While the use of a sub-therapeutic dose of ananti-CD47 agent as a primer agent achieves a desired outcome (e.g., thesubject is “primed” to receive a therapeutically effective dose), thedose is not considered to be a “therapeutic dose” because thesub-therapeutic dose does not effectively increase phagocytosis of atarget cell and is not sufficient to palliate, ameliorate, stabilize,reverse, prevent, slow or delay the progression of the disease state. Asub-therapeutic dose of an anti-CD47 agent can depend on the specificagent used, but is generally less than about 10 mg/kg.

A “maintenance dose” is a dose intended to be a therapeuticallyeffective dose. For example, in experiments to determine thetherapeutically effective dose, multiple different maintenance doses maybe administered to different subjects. As such, some of the maintenancedoses may be therapeutically effective doses and others may besub-therapeutic doses.

Primer agent. As used herein, the term “primer agent” refers to an agentthat primes a subject for a future administration of a therapeuticallyeffective dose of anti-CD47 agent. The inventors have discovered thatwhen a therapeutically effective dose of anti-CD47 agent is administeredto a subject without first administering a primer agent, the highrequired dose can cause a dose-dependent loss of erythrocytes (red bloodcells, RBCs) (e.g., as shown below in mice and non-human primate (NHP)models). One of ordinary skill in the art will readily understand how tomeasure the loss of RBCs. For example, the loss of RBCs can bemonitored, for example, by measuring the percent change in hematocritover time and/or by measuring hemoglobin (e.g., percent change overtime, g/dL, etc.) over time (FIG. 1-FIG. 3 and FIG. 7-FIG. 9). Theseverity of the anemia caused by the loss of RBCs (lethal in some cases)can therefore preclude the use of a therapeutically effective dose ofanti-CD47 agent. However, by administering a primer agent prior toadministering a therapeutically effective dose of anti-CD47 agent, thesubject experiences no adverse effects beyond a temporary, mild anemiathat can occur following administration of the primer agent. Thus,administration of a primer agent serves to prime a subject for a futureadministration of a therapeutically effective dose of anti-CD47.

Subject methods that use a primer agent are particularly relevant whentreating primates because primates are sensitive to RBC count and areprone to develop anemia. Thus, in some embodiments, the subject is aprimate (e.g., human, prosimians, simians, lemurs, lorisoids, tarsiers,monkeys, apes, capuchin monkeys, howler monkeys, squirrel monkeys,baboons, macaques, gibbons, great apes, and the like).

A primer agent increases the number of RBCs in a subject and therebycounteracts the loss of RBCs caused by the administration of atherapeutically effective dose of anti-CD47 agent. Thus, in someembodiments, a primer agent is an erythropoiesis-stimulating agent(ESA). ESAs are known in the art and include, but are not limited toerythropoietin (EPO), EPO derivatives, and EPO-stimulating compounds.Suitable examples include but are not limited to: EPO alpha, EPO beta,EPO delta, EPO omega, EPO zeta, Darbepoetin alfa (Aranesp), Epoetin alfa(Procrit), Epocept (Lupin pharma), Nanokine (Nanogen Pharmaceuticalbiotechnology, Vietnam), Epofit (Intas pharma), Epogen (Amgen), Epogin,Eprex, (Janssen-Cilag), NeoRecormon (Hoffmann-La Roche), Recormon,Methoxy polyethylene glycol-epoetin beta (Mircera)(Roche), Dynepo,Epomax, Silapo (Stade), Retacrit (Hospira), Epocept (LupinPharmaceuticals), EPOTrust (Panacea Biotec Ltd.), Erypro Safe (BioconLtd.), Repoitin (Serum Institute of India Limited), Vintor (EmcurePharmaceuticals), Epofit (Intas pharma), Erykine (IntasBiopharmaceutica), Wepox (Wockhardt Biotech), Espogen (LG lifesciences), ReliPoietin (Reliance Life Sciences), Shanpoietin (ShanthaBiotechnics Ltd.), Zyrop Cadila (Healthcare Ltd.), EPIAO (rHuEPO), and(Shenyang Sunshine Pharmaceutical Co. LTD. China). The dose of ESA thatshould be administered depends on the nature of the agent that is used,and also depends on numerous subject-specific factors (e.g., age,weight, etc.). Methods of determining an appropriate dose of an ESA areknown in the art. In some embodiments, the ESA is administered at a doseaccording to manufacturer's suggestions and in some cases may be as lowas about 50 units/kg, about 100 units/kg or about 150 units/kg of bodyweight or as high as about 17,000 units/kg of body weight.

In some embodiments, the primer agent comprises a sub-therapeutic doseof an anti-CD47 agent. The inventors have discovered that administrationof a sub-therapeutic of an anti-CD47 agent as a primer agent effectivelyprimes the subject for a future administration of a therapeuticallyeffective dose of anti-CD47 agent, preventing severe anemia otherwiseassociated with the therapeutically effective dose.

Accordingly, the term “priming dose” or as used herein refers to a doseof a primer agent (e.g., an anti-CD47 agent, an ESA, etc.) that primes asubject for administration of a therapeutically effective dose ofanti-CD47 agent such that the therapeutically effective dose does notresult in a severe loss of RBCs (reduced hematocrit or reducedhemoglobin). The specific appropriate priming dose of an anti-CD47 agentcan vary depending on the nature of the agent used and on numeroussubject-specific factors (e.g., age, weight, etc.). Examples of suitablepriming doses of an anti-CD47 agent include, but are not necessarilylimited to a range from about 0.05 mg/kg to about 10 mg/kg (e.g., fromabout 0.1 mg/kg to about 10 mg/kg, from about 0.1 mg/kg to about 7.5mg/kg, from about 0.1 mg/kg to about 5 mg/kg, from about 0.1 mg/kg toabout 4 mg/kg, from about 0.1 mg/kg to about 3 mg/kg, from about 0.5mg/kg to about 10 mg/kg, from about 0.5 mg/kg to about 7.5 mg/kg, fromabout 0.5 mg/kg to about 5 mg/kg, from about 0.5 mg/kg to about 4 mg/kg,from about 0.5 mg/kg to about 3 mg/kg, from about 1 mg/kg to about 10mg/kg, from about 1 mg/kg to about 7.5 mg/kg,from about 1 mg/kg to about5 mg/kg, from about 1 mg/kg to about 4 mg/kg, from about 1 mg/kg toabout 3 mg/kg, about 1 mg/kg, about 2 mg/kg, about 3 mg/kg, about 4mg/kg, about 5 mg/kg, about 7.5 mg/kg, or about 10 mg/kg). In someembodiments, the primer agent comprises a combination of an ESA and apriming dose of an anti-CD47 agent.

A “loading dose” is a dose intended to be a priming dose. For example,in experiments to determine an effective priming dose, multipledifferent loading doses may be administered to different subjects. Assuch, some of the loading doses may be priming doses and others may notbe priming doses.

The terms “specific binding,” “specifically binds,” and the like, referto non-covalent or covalent preferential binding to a molecule relativeto other molecules or moieties in a solution or reaction mixture (e.g.,an antibody specifically binds to a particular polypeptide or epitoperelative to other available polypeptides, or binding of a SIRPαpolypeptide). In some embodiments, the affinity of one molecule foranother molecule to which it specifically binds is characterized by aK_(D) (dissociation constant) of 10⁻⁵ M or less (e.g., 10⁻⁶ M or less,10⁻⁷ M or less, 10⁻⁸ M or less, 10⁻⁹ M or less, 10⁻¹⁰ M or less, 10⁻¹¹ Mor less, 10⁻¹² M or less, 10¹³ M or less, 10¹⁴ M or less, 10⁻¹⁵ M orless, or 10⁻¹⁶ M or less). “Affinity” refers to the strength of binding,increased binding affinity being correlated with a lower K_(D).

The term “specific binding member” as used herein refers to a member ofa specific binding pair (i.e., two molecules, usually two differentmolecules, where one of the molecules, e.g., a first specific bindingmember, through non-covalent means specifically binds to the othermolecule, e.g., a second specific binding member). Suitable specificbinding members include agents that specifically bind CD47 and/or SIRPα(i.e., anti-CD47 agents), or that otherwise block the interactionbetween CD47 and SIRPα.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms also apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical mimetic of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers and non-naturally occurring amino acid polymer.

The terms “phagocytic cells” and “phagocytes” are used interchangeablyherein to refer to a cell that is capable of phagocytosis. There arethree main categories of phagocytes: macrophages, mononuclear cells(histiocytes and monocytes); polymorphonuclear leukocytes (neutrophils)and dendritic cells.

The term “sample” with respect to a patient encompasses blood and otherliquid samples of biological origin, solid tissue samples such as abiopsy specimen or tissue cultures or cells derived or isolatedtherefrom and the progeny thereof. The definition also includes samplesthat have been manipulated in any way after their procurement, such asby treatment with reagents; washed; or enrichment for certain cellpopulations, such as cancer cells. The definition also includes samplesthat have been enriched for particular types of molecules, e.g., nucleicacids, polypeptides, etc.

The term “biological sample” encompasses a clinical sample, and alsoincludes tissue obtained by surgical resection, tissue obtained bybiopsy, cells in culture, cell supernatants, cell lysates, tissuesamples, organs, bone marrow, blood, plasma, serum, and the like. A“biological sample” includes a sample comprising target cells or normalcontrol cells or suspected of comprising such cells or biological fluidsderived therefrom (e.g., cancerous cell, infected cell, etc.), e.g., asample comprising polynucleotides and/or polypeptides that is obtainedfrom such cells (e.g., a cell lysate or other cell extract comprisingpolynucleotides and/or polypeptides). A biological sample comprising aninflicted cell from a patient can also include non-inflicted cells.

The term “antibody” is used in the broadest sense and specificallycovers monoclonal antibodies (including full length monoclonalantibodies), polyclonal antibodies, multispecific antibodies (e.g.,bispecific antibodies), and antibody fragments so long as they exhibitthe desired biological activity. “Antibodies” (Abs) and“immunoglobulins” (Igs) are glycoproteins having the same structuralcharacteristics. While antibodies exhibit binding specificity to aspecific antigen, immunoglobulins include both antibodies and otherantibody-like molecules which lack antigen specificity. Polypeptides ofthe latter kind are, for example, produced at low levels by the lymphsystem and at increased levels by myelomas.

“Antibody fragment”, and all grammatical variants thereof, as usedherein are defined as a portion of an intact antibody comprising theantigen binding site or variable region of the intact antibody, whereinthe portion is free of the constant heavy chain domains (i.e. CH2, CH3,and CH4, depending on antibody isotype) of the Fc region of the intactantibody. Examples of antibody fragments include Fab, Fab′, Fab′-SH,F(ab′)₂, and Fv fragments; diabodies; any antibody fragment that is apolypeptide having a primary structure consisting of one uninterruptedsequence of contiguous amino acid residues (referred to herein as a“single-chain antibody fragment” or “single chain polypeptide”),including without limitation (1) single-chain Fv (scFv) molecules (2)single chain polypeptides containing only one light chain variabledomain, or a fragment thereof that contains the three CDRs of the lightchain variable domain, without an associated heavy chain moiety (3)single chain polypeptides containing only one heavy chain variableregion, or a fragment thereof containing the three CDRs of the heavychain variable region, without an associated light chain moiety and (4)nanobodies comprising single Ig domains from non-human species or otherspecific single-domain binding modules; and multispecific or multivalentstructures formed from antibody fragments. In an antibody fragmentcomprising one or more heavy chains, the heavy chain(s) can contain anyconstant domain sequence (e.g. CH1 in the IgG isotype) found in a non-Fcregion of an intact antibody, and/or can contain any hinge regionsequence found in an intact antibody, and/or can contain a leucinezipper sequence fused to or situated in the hinge region sequence or theconstant domain sequence of the heavy chain(s).

As used in this invention, the term “epitope” means any antigenicdeterminant on an antigen to which the paratope of an antibody binds.Epitopic determinants usually consist of chemically active surfacegroupings of molecules such as amino acids or sugar side chains andusually have specific three dimensional structural characteristics, aswell as specific charge characteristics.

Methods

Methods are provided for treating a subject with a therapeutic dose ofanti-CD47 agent. The subject methods include a step of administering aprimer agent to subject, followed by a step of administering atherapeutically effective dose of an anti-CD47 agent to the subject. Insome embodiments, the step of administering a therapeutically effectivedose is performed after at least about 3 days (e.g., at least about 4days, at least about 5 days, at least about 6 days, at least about 7days, at least about 8 days, at least about 9 days, or at least about 10days) after beginning the administration of a primer agent. This periodof time is, for example, sufficient to provide for enhanced reticulocyteproduction by the individual.

In some embodiments, the step of administering a therapeuticallyeffective dose is performed in a range from about 3 days to about 21days (e.g., about 3 days to about 17 days, about 3 days to about 14days, about 3 days to about 12 days, about 4 days to about 12 days,about 5 days to about 12 days, about 5 days to about 11 days, about 5days to about 10 days, about 5 days to about 9 days, about 6 days toabout 8 days, about 3 days, about 4 days, about 5 days, about 6 days,about 7 days, about 8 days, about 9 days, about 10 days, about 11 days,or about 12 days) after beginning the administration of a primer agent.This period of time is, for example, sufficient to provide for enhancedreticulocyte production by the individual.

In some embodiments, two or more primer agents are administered prior toadministering a therapeutically effective dose of an anti-CD47 agent. Insuch cases, the primer agents can be the same agent or can be differentagents. The first primer agent can be administered at the same dose orat a different dose as any subsequently administered primer agent. Insome embodiments, two or more primer agents are administeredsimultaneously and/or the administration of two or primer agents overlapin time, where the administration of one may begin or end before orafter another primer agent.

The administration of a therapeutically effective dose of an anti-CD47agent can be achieved in a number of different ways. In some cases, twoor more therapeutically effective doses are administered after a primeragent is administered. Suitable administration of a therapeuticallyeffective dose can entail administration of a single dose, or can entailadministration of doses daily, semi-weekly, weekly, once every twoweeks, once a month, annually, etc. In some cases, a therapeuticallyeffective dose is administered as two or more doses of escalatingconcentration (i.e., increasing doses), where (i) all of the doses aretherapeutic doses, or where (ii) a sub-therapeutic dose (or two or moresub-therapeutic doses) is initially given and therapeutic doses areachieved by said escalation. As one non-limiting example to illustrateescalating concentration (i.e., increasing doses), a therapeuticallyeffective dose can be administered weekly, beginning with asub-therapeutic dose (e.g., a dose of 5 mg/kg), and each subsequent dosecan be increased by a particular increment (e.g., by 5 mg/kg), or byvariable increments, until a therapeutic dose (e.g., 30 mg/kg) isreached, at which point administration may cease or may continue (e.g.,continued therapeutic doses, e.g., doses of 30 mg/kg). As anothernon-limiting example to illustrate escalating concentration (i.e.,increasing doses), a therapeutically effective dose can be administeredweekly, beginning with a therapeutic dose (e.g., a dose of 10 mg/kg),and each subsequent dose can be increased by a particular increment(e.g., by 10 mg/kg), or by variable increments, until a therapeutic dose(e.g., 30 mg/kg, 100 mg/ml, etc.) is reached, at which pointadministration may cease or may continue (e.g., continued therapeuticdoses, e.g., doses of 30 mg/kg, 100 mg/ml, etc.). In some embodiments,administration of a therapeutically effective dose can be a continuousinfusion and the dose can altered (e.g., escalated) over time.

Dosage and frequency may vary depending on the half-life of theanti-CD47 agent in the patient. It will be understood by one of skill inthe art that such guidelines will be adjusted for the molecular weightof the active agent, e.g. in the use of antibody fragments, in the useof antibody conjugates, in the use of SIRPα reagents, in the use ofsoluble CD47 peptides etc. The dosage may also be varied for localizedadministration, e.g. intranasal, inhalation, etc., or for systemicadministration, e.g. i.m., i.p., i.v., and the like.

Effective administration of primer agent. In some embodiments, a step ofdetermining whether administration of the primer agent was effective isperformed prior to the step of administering a therapeutically effectivedose of an anti-CD47 agent to the subject. If the administration of theprimer agent was not effective, then it may be desirable to begin anewand again administer a primer agent. In such a case, a different doseand/or a different primer agent may be used, or the same dose and sameprimer agent may be used. If the administration of the primer agent waseffective (i.e., the reticulocyte count indicates that theadministration was effective, as described below in more detail), then atherapeutically effective dose of an anti-CD47 agent can be delivered.

Because a priming dose of a primer agent may increase the number of RBCsin a subject (which occurs after administering ESA primer agents as wellas after administering priming doses of anti-CD47 agents), evaluation ofrecently produced (i.e., young) blood cells (i.e., reticulocytes) canserve as an evaluation tool for determining whether administration ofthe primer agent was effective.

Methods of evaluating reticulocytes include measuring the absolute orrelative number of reticulocytes in a blood sample (e.g., a reticulocytecount can be performed on a blood sample from a subject prior to andfollowing administration of a primer agent). Methods to evaluate and/orcount reticulocytes are known by one of ordinary skill in the art andany convenient method may be used. An example of a suitable methodincludes, but is not limited to counting the number of reticulocytes ina sample based on a morphological evaluation. Reticulocytes exhibit amesh-like network that becomes visible with particular stains, e.g., newmethylene blue (NMB). Reticulocytes appear slightly bluer than other redcells when looked at with the normal Romanowsky stain. Reticulocytes arealso slightly larger, which can be picked up as a high MCV (meancorpuscular volume) with a full blood count.

Another example of a suitable method to evaluate reticulocytes includes,but is not limited to counting the number of reticulocytes based on theexpression of a marker of young/immature RBCs (e.g., CD71 expression isincreased in young RBCs relative to older RBCs and CD71 can serve as amarker to identify reticulocytes).

Another example of a suitable method to evaluate reticulocytes includes,but is not limited to counting the number of reticulocytes in a samplebased on measuring the amount of fluorescence exhibited by cells aftercontacting the samples with a fluorescent dye (e.g., thiazole orange,polymethine, etc.) that marks nucleic acid (RNA and DNA), and istherefore a non-selective nucleic acid dye. For example, a non-selectivenucleic acid dye can stain reticulocytes' residual RNA while aDNA-selective dye (e.g., DRAQ5), which can be used in conjunction with anon-selective nucleic acid dye (e.g., thiazole orange), will not stainreticulocytes because reticulocytes have no DNA (reticuloctyes aretherefore DRAQ5 negative). A comparatively middle level of fluorescencedistinguishes reticulocytes from mature RBCs (which have neither RNA norDNA, and therefore very little fluorescence) and from lymphocytes (whichhave a large amount of DNA, unlike reticulocytes). Thus, reticulocytecounts can't be performed in an automated manner (e.g., usingfluorescence-activated cell sorting (FACS)).

Another example of a suitable method to determine whether administrationof the primer agent was effective includes measuring the level of EPO inthe blood. While ESAs can be used to directly stimulate EPO production,priming doses of anti-CD47 agents also cause increases in EPO levels. Assuch, the step of determining whether administration of the primer agentwas effective can comprise measuring the level of EPO in the blood(e.g., prior to and following administration of a primer agent).

Another example of a suitable method to determine whether administrationof the primer agent was effective includes measuring hemoglobin. Methodsto measure hemoblobin are known by one of ordinary skill in the art andany convenient method may be used. Hemoglobin is usually measured as apart of the complete blood count (CBC) from a blood sample. Laboratoryhemoglobin test methods require a blood sample (arterial, venous, orcapillary) and analysis on a hematology analyzer and CO-oximeter.Additionally, noninvasive hemoglobin test methods (e.g., PulseCO-Oximetry) may be used. As one non-limiting example of measuringhemoglobin, red blood cells are broken down to get hemoglobin intosolution. The free hemoglobin is exposed to a chemical containingcyanide, which binds tightly with the hemoglobin molecule to formcyanmethemoglobin. By exposing the sample a specific wavelength of light(e.g., 540 nm) the amount of hemoglobin can be determined.

Determining whether the administration of a primer agent was effective(e.g., via a reticulocyte count) can be performed in a range from about3 days to about 12 days (e.g., about 4 days to about 11 days, about 5days to about 10 days, about 6 days to about 10 days, about 7 days toabout 9 days, about 3 days, about 4 days, about 5 days, about 6 days,about 7 days, about 8 days, about 9 days, about 10 days, about 11 days,or about 12 days) after beginning step (a).

When a reticulocyte count is performed, a count of about 400×10⁹reticulocytes/L or more indicates that the administration of the primeragent was effective. Reticulocyte counts are often expressed as apercentage of red blood cells that are reticulocytes and a normal countfor a healthy adult ranges from 0.5% to 2%. When reticulocyte counts areexpressed in this way, a value of about 4% or more (e.g., 4.5% or more,5% or more, 5.5% or more, or 6 or more) indicates that theadministration of the primer agent was effective. In some cases, afold-increase in reticulocyte number can be calculated and an increaseof 2-fold or more (e.g., 3-fold or more, 3.5-fold or more, 4-fold ormore, 4.5-fold or more, or 5-fold or more) indicates that theadministration of the primer agent was effective. When a hemoglobinmeasurement is performed, an absolute decrease of about 2 to about 4g/dL or a relative decrease of about 12% or more (e.g. about 15% ormore, 17.5% or more, 20% or more ,25% or more, or 30% or more), or arelative decrease ranging from about 12% to about 30% (e.g., about 15%to about 30%, about 15% to about 25%, or about 20% to about 30%)indicates that the administration of the primer agent was effective.

In some embodiments, a subject is monitored for clinical signs ofdisease (e.g., cancer or infection) following administration of atherapeutically effective dose of an anti-CD47 agent.

Kits

Also provided are kits for use in the methods. The subject kits includea primer agent and an anti-CD47 agent. In some embodiments, a kitcomprises two or more primer agents. In some embodiments, a kitcomprises two or more anti-CD47 agents. In some embodiments, a primeragent is provided in a dosage form (e.g., a priming dosage form). Insome embodiments, a primer agent is provided in two or more differentdosage forms (e.g., two or more different priming dosage forms). In someembodiments, an anti-CD47 agent is provided in a dosage form (e.g., atherapeutically effective dosage form). In some embodiments, ananti-CD47 agent is provided in two or more different dosage forms (e.g.,two or more different therapeutically effective dosage forms). In thecontext of a kit, a primer agent and/or an anti-CD47 agent can beprovided in liquid or sold form in any convenient packaging (e.g., stickpack, dose pack, etc.).

In addition to the above components, the subject kits may furtherinclude (in certain embodiments) instructions for practicing the subjectmethods. These instructions may be present in the subject kits in avariety of forms, one or more of which may be present in the kit. Oneform in which these instructions may be present is as printedinformation on a suitable medium or substrate, e.g., a piece or piecesof paper on which the information is printed, in the packaging of thekit, in a package insert, and the like. Yet another form of theseinstructions is a computer readable medium, e.g., diskette, compact disk(CD), flash drive, and the like, on which the information has beenrecorded. Yet another form of these instructions that may be present isa website address which may be used via the internet to access theinformation at a removed site.

Utility. The subject methods and kits can be used to treat anyinfliction where the target cells (e.g., cancer cells, infected cells,etc.) exhibit an increased expression of CD47 relative to normal cellsof the same type. The anti-CD47 agent that is administered inhibits theinteraction between SIRPα (e.g., on a phagocyte) and CD47 on an targetcell (e.g., on a cancer cell, on an infected cell, etc.), therebyincreasing in vivo phagocytosis of the target cell. Subject methodsinclude administering to a subject in need of treatment atherapeutically effective dose of an anti-CD47 agent, including withoutlimitation combinations of the reagent with another drug (e.g., ananti-cancer drug, an anti-infection drug, etc.).

In some embodiments the infliction is a chronic infection, i.e. aninfection that is not cleared by the host immune system within a periodof up to 1 week, 2 weeks, etc. In some cases, chronic infections involveintegration of pathogen genetic elements into the host genome, e.g.retroviruses, lentiviruses, Hepatitis B virus, etc. In other cases,chronic infections, for example certain intracellular bacteria orprotozoan pathogens, result from a pathogen cell residing within a hostcell. Additionally, in some embodiments, the infection is in a latentstage, as with herpes viruses or human papilloma viruses.

Viral pathogens of interest include without limitation, retroviral andlentiviral pathogens, e.g. HIV-1; HIV-2, HTLV, FIV, SIV, etc. HepatitisB virus, etc. Microbes of interest, but not limited to the following,include: Yersinia sp., e.g. Y. pestis, Y. pseudotuberculosis, Yenterocolitica; Franciscella sp.; Pasturella sp.; Vibrio sp., e.g. V.cholerae, V. parahemolyticus; Legionella sp., e.g. L. pneumophila;Listeria sp., e.g. L. monocytogenes; Mycoplasma sp., e.g. M. hominis, M.pneumoniae; Mycobacterium sp., e.g. M. tuberculosis, M. leprae;Rickettsia sp., e.g. R. rickettsii, R. typhi; Chlamydia sp., e.g. C.trachomatis, C. pneumoniae, C. psittaci; Helicobacter sp., e.g. H.pylori, etc. Also included are intracellular protozoan pathogens, e.g.Plasmodium sp, Trypanosoma sp., Giardia sp., Toxoplasma sp., Leishmaniasp., etc.

An infection treated with the methods of the invention generallyinvolves a pathogen with at least a portion of its life-cycle within ahost cell, i.e. an intracellular phase. The methods of the inventionprovide for a more effective removal of infected cells by the phagocyticcells of the host organism, relative to phagocytosis in the absence oftreatment, and thus are directed to the intracellular phase of thepathogen life cycle.

In some embodiments, the methods of the invention involve diagnosis of apatient as suffering from a pathogenic intracellular infection; orselection of a patient previously diagnosed as suffering from apathogenic intracellular infection; treating the patient with a regimenof anti-CD47 therapy, optionally in combination with an additionaltherapy; and monitoring the patient for efficacy of treatment.Monitoring may measure clinical indicia of infection, e.g. fever, whiteblood cell count, etc., and/or direct monitoring for presence of thepathogen.

Treatment may be combined with other active agents. Classes ofantibiotics include penicillins, e.g. penicillin G, penicillin V,methicillin, oxacillin, carbenicillin, nafcillin, ampicillin, etc.;penicillins in combination with β-lactamase inhibitors, cephalosporins,e.g. cefaclor, cefazolin, cefuroxime, moxalactam, etc.; carbapenems;monobactams; aminoglycosides; tetracyclines; macrolides; lincomycins;polymyxins; sulfonamides; quinolones; cloramphenical; metronidazole;spectinomycin; trimethoprim; vancomycin; etc. Cytokines may also beincluded, e.g. interferon γ, tumor necrosis factor α, interleukin 12,etc. Antiviral agents, e.g. acyclovir, gancyclovir, etc., may also beused in treatment.

In some embodiments the infliction is cancer. As noted above, any cancerin which a cancerous cell expresses an increased level of CD47 relativeto a non-cancerous cell of the same type can be treated with the subjectmethods.

The term “cancer”, as used herein, refers to a variety of conditionscaused by the abnormal, uncontrolled growth of cells. Cells capable ofcausing cancer, referred to as “cancer cells”, possess characteristicproperties such as uncontrolled proliferation, immortality, metastaticpotential, rapid growth and proliferation rate, and/or certain typicalmorphological features. A cancer can be detected in any of a number ofways, including, but not limited to, detecting the presence of a tumoror tumors (e.g., by clinical or radiological means), examining cellswithin a tumor or from another biological sample (e.g., from a tissuebiopsy), measuring blood markers indicative of cancer, and detecting agenotype indicative of a cancer. However, a negative result in one ormore of the above detection methods does not necessarily indicate theabsence of cancer, e.g., a patient who has exhibited a complete responseto a cancer treatment may still have a cancer, as evidenced by asubsequent relapse.

The term “cancer” as used herein includes carcinomas, (e.g., carcinomain situ, invasive carcinoma, metastatic carcinoma) and pre-malignantconditions, i.e. neomorphic changes independent of their histologicalorigin. The term “cancer” is not limited to any stage, grade,histomorphological feature, invasiveness, aggressiveness or malignancyof an affected tissue or cell aggregation. In particular stage 0 cancer,stage I cancer, stage II cancer, stage III cancer, stage IV cancer,grade I cancer, grade II cancer, grade III cancer, malignant cancer andprimary carcinomas are included.

Cancers and cancer cells that can be treated include, but are notlimited to, hematological cancers, including leukemia, lymphoma andmyeloma, and solid cancers, including for example tumors of the brain(glioblastomas, medulloblastoma, astrocytoma, oligodendroglioma,ependymomas), carcinomas, e.g. carcinoma of the lung, liver, thyroid,bone, adrenal, spleen, kidney, lymph node, small intestine, pancreas,colon, stomach, breast, endometrium, prostate, testicle, ovary, skin,head and neck, and esophagus.

In an embodiment, the cancer is a hematological cancer. In anembodiment, the hematological cancer is a leukemia. In anotherembodiment, the hematological cancer is a myeloma. In an embodiment, thehematological cancer is a lymphoma.

In an embodiment, the leukemia is selected from acute myeloid leukemia(AML), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia(CLL) and chronic myelogenous leukemia (CML). In an embodiment, theleukemia is AML. In an embodiment, the leukemia is ALL. In anembodiment, the leukemia is CLL. In a further embodiment, the leukemiais CML. In an embodiment, the cancer cell is a leukemic cell, forexample, but not limited to, an AML cell, an ALL cell, a CLL cell or aCML cell.

Suitable cancers responsive to treatment using an anti-CD47 agentinclude without limitation leukemia; acute myeloid leukemia (AML); acutelymphoblastic leukemia (ALL); metastasis; minimal residual disease;solid tumor cancers, e.g., breast, bladder, colon, ovarian,glioblastoma, leiomyosarcoma, and head & neck squamous cell carcinomas;etc. For examples, see: (i) Willingham et al., Proc Natl Acad Sci USA.2012 Apr. 24; 109(17):6662-7: “The CD47-signal regulatory protein alpha(SIRPα) interaction is a therapeutic target for human solid tumors”;(ii) Edris et al., Proc Natl Acad Sci USA. 2012 Apr 24;109(17):6656-61:“Antibody therapy targeting the CD47 protein is effective in a model ofaggressive metastatic leiomyosarcoma”; and (iii) US patent application20110014119; all of which are herein incorporated in their entirety.

Pharmaceutical Compositions. Suitable anti-CD47 agents and/or primeragents can be provided in pharmaceutical compositions suitable fortherapeutic use, e.g. for human treatment. In some embodiments,pharmaceutical compositions of the present invention include one or moretherapeutic entities of the present invention or pharmaceuticallyacceptable salts, esters or solvates thereof. In some other embodiments,the use of an anti-CD47 agent or primer agent includes use incombination with another therapeutic agent (e.g., another anti-infectionagent or another anti-cancer agent). Therapeutic formulations comprisingone or more anti-CD47 agents and/or primer agents of the invention areprepared for storage by mixing the anti-CD47 agent or primer agenthaving the desired degree of purity with optional physiologicallyacceptable carriers, excipients or stabilizers (Remington'sPharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the formof lyophilized formulations or aqueous solutions. The anti-CD47 agent orprimer agent composition will be formulated, dosed, and administered ina fashion consistent with good medical practice. Factors forconsideration in this context include the particular disorder beingtreated, the particular mammal being treated, the clinical condition ofthe individual patient, the cause of the disorder, the site of deliveryof the agent, the method of administration, the scheduling ofadministration, and other factors known to medical practitioners.

The anti-CD47 agent or primer agent can be administered by any suitablemeans, including topical, oral, parenteral, intrapulmonary, andintranasal. Parenteral infusions include intramuscular, intravenous(bollus or slow drip), intraarterial, intraperitoneal, intrathecal orsubcutaneous administration.

The anti-CD47 agent or primer agent need not be, but is optionallyformulated with one or more agents that potentiate activity, or thatotherwise increase the therapeutic effect. These are generally used inthe same dosages and with administration routes as used hereinbefore orabout from 1 to 99% of the heretofore employed dosages.

An anti-CD47 agent or primer agent is often administered as apharmaceutical composition comprising an active therapeutic agent andanother pharmaceutically acceptable excipient. The preferred formdepends on the intended mode of administration and therapeuticapplication. The compositions can also include, depending on theformulation desired, pharmaceutically-acceptable, non-toxic carriers ordiluents, which are defined as vehicles commonly used to formulatepharmaceutical compositions for animal or human administration. Thediluent is selected so as not to affect the biological activity of thecombination. Examples of such diluents are distilled water,physiological phosphate-buffered saline, Ringer's solutions, dextrosesolution, and Hank's solution. In addition, the pharmaceuticalcomposition or formulation may also include other carriers, adjuvants,or nontoxic, nontherapeutic, nonimmunogenic stabilizers and the like.

In still some other embodiments, pharmaceutical compositions can alsoinclude large, slowly metabolized macromolecules such as proteins,polysaccharides such as chitosan, polylactic acids, polyglycolic acidsand copolymers (such as latex functionalized Sepharose™, agarose,cellulose, and the like), polymeric amino acids, amino acid copolymers,and lipid aggregates (such as oil droplets or liposomes).

A carrier may bear the agents in a variety of ways, including covalentbonding either directly or via a linker group, and non-covalentassociations. Suitable covalent-bond carriers include proteins such asalbumins, peptides, and polysaccharides such as aminodextran, each ofwhich have multiple sites for the attachment of moieties. A carrier mayalso bear an anti-CD47 agent or primer agent by non-covalentassociations, such as non-covalent bonding or by encapsulation. Thenature of the carrier can be either soluble or insoluble for purposes ofthe invention. Those skilled in the art will know of other suitablecarriers for binding anti-CD47 agents and/or primer agents, or will beable to ascertain such, using routine experimentation.

Acceptable carriers, excipients, or stabilizers are non-toxic torecipients at the dosages and concentrations employed, and includebuffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid and methionine; preservatives (suchas octadecyidimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionicsurfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).Formulations to be used for in vivo administration must be sterile. Thisis readily accomplished by filtration through sterile filtrationmembranes.

The active ingredients may also be entrapped in microcapsule prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsule and poly-(methylmethacylate) microcapsule,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

Carriers and linkers specific for radionuclide agents includeradiohalogenated small molecules and chelating compounds. A radionuclidechelate may be formed from chelating compounds that include thosecontaining nitrogen and sulfur atoms as the donor atoms for binding themetal, or metal oxide, radionuclide.

Radiographic moieties for use as imaging moieties in the presentinvention include compounds and chelates with relatively large atoms,such as gold, iridium, technetium, barium, thallium, iodine, and theirisotopes. It is preferred that less toxic radiographic imaging moieties,such as iodine or iodine isotopes, be utilized in the methods of theinvention. Such moieties may be conjugated to the anti-CD47 agent orprimer agent through an acceptable chemical linker or chelation carrier.Positron emitting moieties for use in the present invention include ¹⁸F,which can be easily conjugated by a fluorination reaction with theanti-CD47 agent or primer agent.

Typically, compositions are prepared as injectables, either as liquidsolutions or suspensions; solid forms suitable for solution in, orsuspension in, liquid vehicles prior to injection can also be prepared.The preparation also can be emulsified or encapsulated in liposomes ormicro particles such as polylactide, polyglycolide, or copolymer forenhanced adjuvant effect, as discussed above. Langer, Science 249: 1527,1990 and Hanes, Advanced Drug Delivery Reviews 28: 97-119, 1997. Theagents of this invention can be administered in the form of a depotinjection or implant preparation which can be formulated in such amanner as to permit a sustained or pulsatile release of the activeingredient. The pharmaceutical compositions are generally formulated assterile, substantially isotonic and in full compliance with all GoodManufacturing Practice (GMP) regulations of the U.S. Food and DrugAdministration.

Toxicity of the anti-CD47 agents and/or primer agents can be determinedby standard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., by determining the LD₅₀ (the dose lethal to 50% of thepopulation) or the LD₁₀₀ (the dose lethal to 100% of the population).The dose ratio between toxic and therapeutic effect is the therapeuticindex. The data obtained from these cell culture assays and animalstudies can be used in further optimizing a therapeutic dosage rangeand/or a priming dosage range for use in humans. The exact formulation,route of administration and dosage can be chosen by the individualphysician in view of the patient's condition.

The invention now being fully described, it will be apparent to one ofordinary skill in the art that various changes and modifications can bemade without departing from the spirit or scope of the invention.

EXPERIMENTAL

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Centigrade,and pressure is at or near atmospheric.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference.

The present invention has been described in terms of particularembodiments found or proposed by the present inventor to comprisepreferred modes for the practice of the invention. It will beappreciated by those of skill in the art that, in light of the presentdisclosure, numerous modifications and changes can be made in theparticular embodiments exemplified without departing from the intendedscope of the invention. For example, due to codon redundancy, changescan be made in the underlying DNA sequence without affecting the proteinsequence. Moreover, due to biological functional equivalencyconsiderations, changes can be made in protein structure withoutaffecting the biological action in kind or amount. All suchmodifications are intended to be included within the scope of theappended claims.

EXAMPLES Example 1

Single Injection of Anti-CD47 Antibodies in Wild Type Mice

A single 250 μg IP injection of MIAP410 (IgG1 isotype) or MIAP470 (IgG2aisotype) was administered to wild type mice. This antibody dose isroughly equivalent to a 10 mg/kg dose. Blood was collected fromretro-orbital plexus and CBC analysis was performed on HemaTruehematology analyzer 1,3, and 6 days after antibody injection. Thepercent change in hematocrit (HCT) and RBC values are shown in FIG. 1.All anti-CD47 mAb treated mice developed anemia, the nadir of whichoccurred about 3 days after injection. The MIAP470 caused a moresignificant anemia, which is likely due to differential FcR mediatedeffects caused by the IgG1 and IgG2a isotypes.

Single Injection of Anti-CD47 Antibodies in CD47^(−/−) Mice

CD47^(−/−) mice were acquired from Jackson Laboratory and injected IPwith 250 μg of control mouse IgG, MIAP410, or MIAP470. Blood wascollected and analyzed 72 hours after mAb injection. No anemia wasobserved in any of the injected CD47^(−/−) mice. The percent change inhemoglobin is shown in FIG. 2. This demonstrates that the observedanemia in wild type mice was a direct result of anti-CD47 antibodiesbinding CD47.

Multiple Injections of Anti-CD47 Antibodies in Wild Type Mice

WT mice were given an IP injection of 250 μg of control mouse IgG,MIAP410, or MIAP740 every 3 days (represented by dashed vertical line inFIG. 3). Erythrocyte toxicity was monitored by CBC analysis prior toeach injection. An acute drop in HCT occurred upon the first antibodyinjection (Day 0). The second injection (on day 3) did not result in afurther drop in hematocrit. Mice appeared to become resistant tosubsequent injections and eventually returned to a range similar to thatof control IgG treated mice. The discovery that repeated administrationof anti-CD47 antibodies does not exacerbate the initial anemia is thebasis of the subsequent experiments in non-human primates.

Example 2

Verification of Dosing Strategy in Non-human Primates (NHPs)

The Hu5F9-G4 anti-CD47antibody (and its parent 5F9) binds human CD47,but does not bind mouse CD47. In order to identify an appropriatetoxicology species, the sequence of macaque (cynomolgus) non-humanprimate (NHP) CD47 was aligned with human CD47 and it was determinedthat the two sequences contain only 3 amino acid differences in theextracellular domain (FIG. 4). All 3 non-conserved amino acids arelocated outside of the SIRP-alpha interacting region as has beendetermined by published X-ray crystal structures.

A cynomolgus NHP CD47-Fc fusion protein was generated and it wasdetermined that

Hu5F9-G4 does in fact bind to cynomolgus NHP CD47 (FIG. 5). Surfaceplasmon resonance (SPR) affinity measurements were further conducted byusing Biacore. The results show that Hu5F9-G4 binds cynomolgus CD47 withan affinity comparable to that of human CD47 (FIG. 6).

In addition, flow cytometry and immunofluorescence, respectively, wereused to show that Hu5F9-G4 antibody binds cynomolgus NHP leukocytes andnormal tissues in a distribution similar to human leukocytes andtissues. Taken together, these studies demonstrated that the cynomolgusmonkey is a relevant species for the safety and toxicology studies.

Anti-CD47 Antibodies Causes Dose Dependent Anemia in NHPs (Single Dose)

A number of NHP studies were conducted using purified Hu5F9-G4antibodies produced by Lonza using their proprietary GlutamineSynthetase (GS) expression system. Hu5F9-G4 was administered as a singledose at 0.1, 0.3, 1, 3, 10, or 30 mg/kg and clinical pathologyparameters were monitored, including complete blood counts and metabolicpanels. A dose-dependent anemia, associated with reticulocytosis andspherocytosis, was observed without hepatic or renal dysfunction (FIG.7). There was no free plasma hemoglobin detected, indicating the absenceof intravascular hemolysis. Monitoring of pharmacokinetics throughmeasurement of serum levels indicated that there is a large antigen sinkresulting in a short half-life (FIG. 7). With single doses, only 10 and30 mg/kg were able to transiently achieve serum levels in the rangeassociated with efficacy in xenograft studies. Thus, proper dosing canachieve and maintain therapeutically effective anti-CD47 agent levelswhile minimizing anemia.

Escalating Concentrations of Anti-CD47 Antibodies do not ExacerbateAnemia

We speculated that prior administration of erythropoietin (EPO) wouldblunt the anemia by stimulating young RBC production. From theseconsiderations, we conducted a separate dose-escalation study in NHPbased on the hypothesis that initial low doses would blunt the loss ofaged RBC and stimulate production of less-susceptible young RBC, therebyfacilitating tolerance of subsequent larger doses (FIG. 8). Two animalswere enrolled into this study and dosed at one week intervals: one withEPO pre-treatment (3, 10, 30, 100, and 300 mg/kg), and one with nopre-treatment (1, 3, 10, 30, and 100 mg/kg). In both cases, the NHPexhibited a mild anemia with initial dosing that did not worsen withrepeated administrations. In fact, the hemoglobin only reached the upperthreshold for transfusion in humans, even without EPO pretreatment. Theanimals tolerated all doses well, including 100 and 300 mg/kg, with noadditional blood or metabolic abnormalities. At the end of the study,both animals were euthanized, and necropsy and histopathology analysisrevealed no abnormalities.

From this dose-escalation study, we determined the pharmacokinetics ofHu5F9-G4 in NHP. Consistent with the presence of a large antigen sink ofCD47 expressed by normal tissues, the initial low doses of Hu5F9-G4 wererapidly cleared from the serum with a half-life of 24 hours (FIG. 9). Incontrast, the higher doses of Hu5F9-G4 produced sustained serum levelsindicating saturation of the antigen sink. The animal dosed at 300 mg/kghad a peak level of 5 mg/ml with a sustained level of more than 1 mg/mlfor nearly 2 weeks.

Single Loading Dose of Anti-CD47 Antibodies Enables Higher MaintenanceDoses

These studies demonstrated that Hu5F9-G4 can be administered to NHP atdoses capable of achieving prolonged, therapeutic serum levels withoutmajor toxic effects. To model potential clinical dosing strategies, wehave conducted another NHP study using a loading-maintenance dosingapproach. In this study, Hu5F9-G4 is administered with a loading dosecapable of stimulating mild anemia and reticulocytosis without grade 3toxicity. Our single dose data (See above), led us to select either 1 or3 mg/kg on day 1 as the loading dose. One week later, a maintenance doseof either 10 or 30 mg/kg is administered, and continued weekly for 3doses. Both loading doses mitigate the severity of the anemia, even withthe 30 mg/kg maintenance doses, and no grade 3 toxicity develops. PKdata following the first maintenance dose indicate that the animals haveachieved therapeutic levels. This study suggests that aloading-maintenance strategy mitigates anemia (preventing grade 3toxicity) while achieving potentially therapeutic drug levels. FIG. 10presents reticulocyte count data demonstrating the level ofreticulocytosis associated with various doses of an anti-CD47 agent(hu5F9-G4 antibody in this case).

Example 3

Therapeutic Efficacy of anti-CD47 Antibodies in XenotransplantationAssays

We previously reported preclinical evidence that a blocking anti-CD47monoclonal antibody (clone B6H12, mouse IgG1) was effective atinhibiting the growth and metastasis of solid tumors, including breast,bladder, colon, ovarian, glioblastoma, leiomyosarcoma, and head & necksquamous cell carcinoma (PMID: 22451913, 22451919). Anti-CD47 monoclonalantibodies also caused similar inibition of hematologic tumor growth(PMID: 21177380, 20813259, 19632179, 19632178). We have now confirmedthat humanized antibodies (e.g., the humanized anti-CD47 antibody usedin the studies above, hu5F9-G4) is also highly effective at inhibitingsolid tumor growth and eliminating metastases (FIG. 11).

To investigate the efficacy of hu5F9-G4 on solid tumors, a human bladdercancer cell line (639V) was subcutaneously transplanted intoimmunodeficient mice. Tumor bearing mice were treated with PBS orhu5F9-G4. Due to tumor burden, all PBS-treated mice were euthanizedafter 4 weeks of treatment. In contrast, no tumor growth was observed inthe hu5F9-G4 treated cohort (FIG. 11). These mice we then monitored foran additional 4 weeks (without further hu5F9-G4 treatment). No tumorgrowth was observed in mice that had been treated hu5F9-G4, indicatingthat the tumors had been fully eliminated (FIG. 11A).

To evaluate the effect of hu5F9-G4 on formation of tumor metastases, wesubcutaneously engrafted a human metastatic prostate tumor specimen intoimmunodeficient mice. Upon tumor engraftment, we initiated treatmentwith PBS or hu5F9-G4. After 6 weeks of treatment, a significant decreasein the number and size of lymph node metastases was observed (FIG. 11B).These results indicate that hu5F9-G4 can inhibit or eliminate tumormetastases.

To demonstrate the potential of hu5F9-G4 to eliminate establishedmetastases, we engrafted primary human breast cancer cells into themouse mammary fat pad. After confirming the presence of tumor metastasesin the lungs, we resected the primary tumor and initiated treatment withPBS or hu5F9-G4. After 4 weeks, a significant inhibition of lungmetastasis growth was observed in hu5F9-G4 treated mice (FIG. 12A).Moreover, a complete elimination of tumor metastases in the brain wasobserved (FIG. 12B). Hu5F9-G4 also inhibited the regrowth of theresected primary tumor, indicating that hu5F9-G4 can also be effectivein treating minimal residual disease (FIG. 12C).

Serum concentrations of hu5F9-G4 were monitored throughout theexperiment. Hu5F9-G4 concentrations between 100-200 μg/ml wereassociated with therapeutic efficacy (FIG. 12AD). These data demonstratethat humanized antibodies (e.g., hu5F9-G4), have the same generalproperties related to the treatment of disease (e.g., cancer or chronicinfection) as the non-humanized antibodies and the subject methods willbe effective when using a humanized antibody to treat cancer and/or totreat chronic infection.

Example 4

In previous toxicology experiments in cynomolgus monkeys, a singleinjection of our humanized anti-CD47 monoclonal antibody (Hu5F9-G4)produced unacceptable levels of anemia (Hb>7 g/ dL) when dosed at orabove 10 mg/kg. Hu5F9-G4 doses less than 10 mg/kg are insufficient toproduce serum levels associated with therapeutic efficacy in ourpreclinical models (100-200 ug/ml) This new study determined if a single“priming dose” of 5 mg/kg was sufficient to protect cynomologus monkeysfrom subsequent “maintenance doses” at levels that would otherwise betoxic (and probably lethal) (See study design: FIG. 13).

Despite the anemia related to administration of Hu5F9-G4, no evidence oftoxicity was observed in clinical signs in any animal, and thus, thepriming/maintenance dosing strategy allows Hu5F9-G4 to be clinicallywell-tolerated, even at doses as high as 300 mg/kg (FIG. 14). We believethat the administration of Hu5F9-G4 accelerates the process ofelimination of aging RBCs by substituting gradual loss of CD47 withimmediate blockade of CD47 on aging RBCs. The premature loss of agingRBCs is compensated by an ensuing reticulocytosis (which was observedacross all studies), and over time, the initial anemia resolves as theaged RBCs are replaced with younger cells, and as a result, the agedistribution of the RBC pool is shifted to younger cells. Serumconcentrations in Groups 2-4 (FIG. 13, FIG. 14, FIG. 15) were well abovethe minimum 100-200 ug/ml level associated with therapeutic efficacy.

FIG. 13. Study design. The animals (# males and females in far rightcolumn) were divided into 5 groups, one of which did not receiveanti-CD47 antibody. All animals in Groups 2-5 received a priming dose (5mg/kg) and Groups 2-5 received the listed maintenance doses thereafter.

FIG. 14. Hemoglobin levels in all cohorts over the duration of thestudy. The horizontal dashed line represents a dose limiting toxicity inthis study (Hb<7).Each dot on this graph represents an individualanimal. There is no statistical difference in the Hb levels at thedifferent maintenance dose levels. The priming dose (5 mg/kg) protectsagainst subsequent doses no matter how high.

FIG. 15. Pharmacokinetic profile of Hu5F9-G4 in all cohorts. Groups 3-4achieve serum concentrations of Hu5F9-G4 well above minimumconcentrations associated with efficacy (Target Range).

Example 5

Studies were conducted in rhesus and/or cynomolgus monkeys, both ofwhich are considered to be pharmacologically relevant animal species(CD47 of rhesus monkeys shares 100% sequence homology with CD47 ofcynomolgus monkeys).

All studies were conducted in compliance with United States Food andDrug Administration (FDA) Good Laboratory Practice (GLP) Regulations (21CFR Part 58). Development of Hu5F9-G4 also followed applicable ICH,Committee on Proprietary, and FDA guidance documents.

In Vitro Hemolysis Assay of Hu5F9-G4 (Humanized Anti-CD47 Antibody)

Because CD47 is expressed on red blood cells and functions in theclearance of aging red blood cells, this study was conducted to evaluateif Hu5F9-G4 caused direct intravascular hemolysis of either human ormonkey (rhesus and cynomolgus) red blood cells, using free hemoglobin asa read out. Hu5F9-G4 did not cause hemolysis of either human or monkey(rhesus or cynomolgus) red blood cells.

Cytokine Production in Human PBMC Stimulated with Hu5F9-G4 In Vitro

The objective of this study was to evaluate the potential induction ofcytokine release by Hu5F9-G4 in human peripheral blood mononuclear cells(PBMCs). In this in vitro study, cultured PMBCs collected from threeseparate donors were incubated with either Hu5F9-G4 (20 μg/mL platebound) or a non-specific human IgG4 antibody (negative control) oranti-CD3/anti-CD28 antibodies (positive control). Fifty differentcytokines, including many pro-inflammatory cytokines associated withcytokine storm (e.g., TNF-α, IL-1, IL-4, IL-6) were evaluated by Luminexmultiplex analysis. Of the different cytokines evaluated, Hu5F9-G4 didnot induce cytokine release in human PBMCs. In addition, no clinicalsigns of cytokine release syndrome were observed in any of the monkeystudies. Treatment of human PBMCs with Hu5F9-G4 did result in areduction of levels of IL1-RA, MIP1b/CCL4, IL-8, and ENA-78/CXCL5 incomparison to PBMCs treated with the non-specific IgG4 antibody or theCD3/CD28 antibodies. While the relationship between CD47 and these fourcytokines is not clear, no evidence of any treatment-related effectsthat could be directly associated with a reduction in cytokine levelswas observed in the monkey studies.

Single Dose Study of Hu5F9-G4 and B6H12-G4 Administered by 1-HourIntravenous Infusion or Subcutaneous Injection to Cynomolgus Monkeys

The purpose of this study was to evaluate the potential toxicity andtoxicokinetics of

Hu5F9-G4 administered as a single dose by a 1-hour IV infusion to malecynomolgus monkeys. In this study, Hu5F9-G4 was administered at a doseof 10 mg/kg to a single male monkey on Day 1. The monkey was evaluatedfor changes in clinical signs, food consumption, body weight, andclinical pathology parameters (hematology, coagulation, hematology,clinical chemistry). Samples were collected for toxicokinetic analysisthroughout the duration of the study; the animal was returned to thetesting facility animal colony on Day 14.

Treatment-related changes were observed in hematology parameters andincluded mild to moderate decreases in red blood cell count (RBC),hemoglobin, hematocrit, markedly increased reticulocyte counts and redcell distribution width, and transient increases in white blood cell,lymphocyte, and monocyte counts. Changes were also observed in clinicalchemistry parameters that were considered likely related to Hu5F9-G4,which included transient increases in lactate dehydrogenase, bilirubin,AST, and ALT. Changes in hematology and clinical chemistry parameterspartially or completely returned to baseline levels by Day 14.

In summary, a single administration of Hu5F9-G4 at 10 mg/kg was welltolerated, with treatment-related findings limited to transient changesin hematology and clinical chemistry parameters.

Study Via Intravenous Infusion Administration to Rhesus Monkey

This study was conducted to evaluate the potential effects of Hu5F9-G4on hematology and clinical chemistry parameters when administered torhesus monkeys at the same testing facility where the studies incynomolgus monkeys were conducted (Charles River Laboratories, Reno,Nev.). In this study, Hu5F9-G4 was administered as a single dose tofemale rhesus monkeys (N=2) via a 1-hour IV infusion at 3 mg/kg. Theanimals were evaluated for 14 days, and then returned to the facilityanimal colony.

Administration of HuF59-G4 was well tolerated, and no changes considereddirectly related to Hu5F9-G4 were noted in clinical signs, body weights,or food consumption. Watery feces were observed in both animals on Days7, 8, and 14, which was likely related to study-related proceduresrather than a direct effect of Hu5F9-G4. In addition, watery feces werenot reported in any other monkey study. Treatment-related changes wereobserved in both animals in hematology parameters, including decreasesin RBC and hemoglobin levels; however, these decreases recovered by Day14 and were not severe with nadirs of 9.4 and 9.1 g/dL (Table 1). Freeplasma hemoglobin was not detected in either animal at any time point.An increase in total bilirubin was also observed in each animal, butconsistent with the hematology changes, showed a continued trend forreversibility to the end of the study.

In summary, Hu5F9-G4 was well tolerated in rhesus monkeys, and thetreatment-related changes observed in this study were consistent withthe findings noted in the cynomolgus monkey studies conducted at CharlesRiver Laboratories (Reno, Nev.).

TABLE 1 Changes in Clinical Pathology Parameters in Rhesus MonkeysAnimal RBC HGB Total Bilirubin No. Study Day (10⁶/μl) (g/dL) (mg/dL) 700Pre-study 6.12 13.8 0.18 1 (8 hr post-dose) 5.14 11.7 1.30 1 (24 hrpost-dose) 4.9 11.3 1.69 2 4.36 10.0 0.98 3 4.18 9.8 0.56 5 4.04 9.40.34 7 4.11 9.9 0.49 10 4.25 10.8 0.43 14 4.62 11.4 0.32 701 Pre-study5.47 12.7 0.12 1 (8 hr post-dose) 4.85 11.5 1.89 1 (24 hr post-dose)4.65 10.8 0.77 2 4.21 9.9 0.44 3 4.02 9.8 0.34 5 4.01 9.6 0.33 7 3.89*9.1 0.41 10 4.15 10.4 0.41 14 4.9 12.7 0.23Pharmacokinetic and Tolerability Study of Hu5F9-G4 Administered toRhesus Monkeys

The initial purpose of this study was to evaluate the pharmacokineticsand potential effects of Hu5F9-G4 administered as a 1-hour IV infusionor by intrathecal administration in rhesus monkeys implanted with anintrathecal reservoir. However, due to the severe anemia observed in thefirst monkey administered Hu5F9-G4 via a 1-hour IV infusion, theremaining components of the study, including the intrathecaladministration phase, were abandoned. In this study, one male rhesusmonkey was administered Hu5F9-G4 via a 1-hour IV infusion as a 3 mg/kgpriming dose on Day 0, followed by a 1 mg/kg maintenance doseadministered on Days 15 and 22 (the maintenance dose in the initialstudy design was 30 mg/kg administered on Days 8, 15, 22, and 29).

A substantial reduction in RBC counts and hemoglobin was observed within24 hours following administration of the 3 mg/kg priming dose (Table 2).Due to the anemia observed in this animal, the first scheduledmaintenance dose on Day 8 was not administered. The RBC counts andhemoglobin levels showed a trend to recovery, and by Day 14, the RBCcounts and hemoglobin levels had return to 4.31 M/μL and 10.8 g/dL,respectively. Dosing was therefore resumed on Day 14; however, themaintenance dose was reduced to 1 mg/kg (rather than 30 mg/kg). On Day16, the RBC counts and hemoglobin again began to decrease, however, byDay 17, both RBC counts and hemoglobin began to recover. The animal wasthen administered a second maintenance dose on Day 21 (however, noadditional clinical pathology data was collected after Day 21). Inaddition, while a reduction in platelets was noted two days afteradministration of the first maintenance dose (on Day 14), the plateletsreturn to pre-study levels by Day 21; it is unclear at this time if thereduction in platelets noted in this animal was directly related toHu5F9-G4 since this change was not observed in any other monkey studies,including the single dose rhesus monkey study.

TABLE 2 Changes in Hematology Parameters in a Rhesus Monkey AdministeredHu5F9-G4 RBC HGB Study Day (M/μL) (g/dL) Platelets (K/μL) Pre-study 5.0912.2 548  0 (8 hr post-dose) 3.73 7.8 380  1 3.86 8.0 411  2 3.42 7.2416  3 3.27 7.0 517  6 3.31 7.8 661  9 3.56 9.1 632 13 3.94 8.9 373 14(pre-dose) 4.31 10.8 374 15 3.87 8.3 146 16 3.95 8.2 130 17 4.19 8.5 19720 4.01 8.7 494 21 (pre-dose) 4.37 9.1 622A Single Dose Study and Repeat Dose Study of Hu5F9-G4 and Single DoseStudy of FD6-IgG2 Administered by 1-Hour Intravenous Infusion toCynomolgus Monkeys

The anemia observed in a previous single-dose study may be related tothe pharmacological action of Hu5F9-G4 as a result of binding CD47expressed on RBCs. As RBCs age, they gradually lose CD47 expression,lose sialic acids from glycoproteins and glycolipids, and reorganizemembrane phospholipids in a manner that presumably accumulatespro-phagocytic signals, leading to their elimination by phagocytosis(Danon, 1988). We hypothesize that administration of Hu5F9-G4accelerates the process of aging RBC elimination by substituting gradualloss of CD47 with immediate blockade of CD47 on aging RBCs. Thepremature loss of aging RBCs is compensated by an ensuingreticulocytosis, and the initial anemia resolves as aged RBCs arereplaced with younger RBCs and the age distribution of the RBC pool isshifted to younger cells. Based on these considerations, this study wasconducted to assess whether i) initial low doses of Hu5F9-G4 might causea limited loss of aged RBC that is nevertheless sufficient to induce areticulocytosis and thereby stimulate production of less-susceptibleyoung RBC and protect the animal from serious anemia; and ii)pre-treatment with Erythropoietin (EPO; an erythropoiesis stimulatingagent that stimulates RBC production) may induce production ofless-susceptible young RBC, thereby compensating for the clearance ofaged RBC following Hu5F9-G4 administration. Another antibody candidate(FD6-IgG2) was evaluated in this study but will not be discussed in thisIND. In this study, male cynomolgus monkeys were administered Hu5F9-G4by a 1-hour IV infusion as a single-dose at 1 mg/kg or once weekly at 3mg/kg for 4 weeks (Days 1, 8, 15, 22). One monkey administered onceweekly doses of Hu5F9-G4 was pre-treated with Erythropoietin (EPO) by IVinjection (17,000 U/kg) on Day -5 to assess if pretreatment with EPOwould reduce the Hu5F9-G4-related anemia previously observed. Inaddition, one monkey administered once weekly doses of Hu5F9-G4 was alsoadministered Dexamethasone by IV injection (0.5 mg/kg) and Benadryl byintramuscular (IM) injection (5 mg/kg) on Days 1, 2, 5, 8, 9, 12, 15,16, 19, 22, 23, and 26 (Dexamethasone and Benadryl were administered atthe same time) to evaluate if administration of Dexamethasone andBenadryl would reduce the Hu5F9-G4-related anemia. The study design ispresented in Table 3.

TABLE 3 Single and Repeat Doses of Hu5F9-G4 Administered to CynomolgusMonkeys Hu5F9-G4 EPO Hu5F9-G4 Dose Dexamethasone/Benadryl Dose Group^(A)(mg/kg) Days Dose Days^(B) Days^(C) 1 1 1 — — 2 3 1, 8, 15, 1, 2, 5, 8,9, 12, 15, 16, 19, 22 22, 23, 26, 3 3 1, 8, 15, — −5 22 4 3 1, 8, 15, 22— — ^(A)Group 1 was released to the animal colony on Day 31; Groups 2and 4 were terminated on Day 31 and Group 3 was terminated on Day 29^(B)Dexamethasone (IV injection; 0.5 mg/kg) and Benadryl (IM injection;5 mg/kg) were administered at same time on Days 1, 8, 15, 22 60 minutesprior to Hu5F9-G4 ^(C)EPO was administered IV at 17,000 U/kg

Standard safety parameters (e.g., clinical observations, body weights,clinical pathology, etc.) were incorporated in the study, and becauseCD47 is expressed in the brain (add reference), veterinary neurologicexaminations were performed. Blood was collected at time points acrossthe study for toxicokinetics. Animals in Groups 2 and 3 were terminatedon Day 31, and the monkey in Group 4 was terminated on Day 29 (themonkey in Group 1 was returned to the Test Facility animal colony).

No unscheduled deaths occurred and no treatment-related changes werenoted in clinical signs, body weights, food consumption, veterinaryneurologic examinations, coagulation or urinalysis parameters, organweights, or macroscopic or microscopic examinations.

Changes related to Hu5F9-G4 were limited to changes in hematology andclinical chemistry parameters. Changes in the single-dose animal(Group 1) included decreased red cell mass (RBC count, hemoglobin, andhematocrit levels) and mean corpuscular volume (MCV), and increased meancorpuscular hemoglobin concentration (MCHC) and red cell distributionwidth (RDVV); these changes were observed by Day 2. This animal also hada robust reticulocyte response, and changes in RBC morphology observedin blood smear evaluations included minimal to mild spherocytes,anisosytosis, and polychromasia. Treatment-related changes in monkeysadministered Hu5F9-G4 once weekly included decreases in RBC mass and MCVand increases in MCHC. These changes were noted by Day 3 and were lesspronounced in the animal pretreated with EPO compared to the othermonkeys administered Hu5F9-G4 alone or with Dexamethasone/Benadryl. Thechanges in RBC mass partially recovered by Day 27 (5 days after the lastdose on Day 22), and were associated with a corresponding robustreticulocytes response. Similar to the single-dose monkey, changes inRBC morphology included minimal to moderate spherocytes, anisosytosis,and polychromasia. Meaningful elevations in free plasma hemoglobin werenot detected in the single-dose or repeat-dose Hu5F9-G4-treated monkeys.

Changes in clinical chemistry parameters considered related to Hu5F9-G4administration included increased lactate dehydrogenase, aspartateaminotransferase (single-dose animal only), total bilirubin (minimal),and decreased haptoglobin (repeat-dose treated Hu5F9-G4 alone andDexamethasone/Benadryl monkeys only). These changes in clinicalchemistry parameters showed evidence of complete or partial recovery bythe end of the study.

In summary, administration of Hu5F9-G4 as a single dose at 1 mg/kg oronce weekly doses for 4 weeks at a dose of 3 mg/kg (alone or withpretreatment with EPO or in combination with administration ofDexamethasone/Benadryl) was well-tolerated in male cynomolgus monkeys,and treatment related effects were limited to changes in hematology(including RBC morphology) and clinical chemistry parameters. Partial orcomplete recovery was noted in changes in RBC mass and clinicalchemistry parameters by the end of study.

Repeat Dose Study of Hu5F9-G4 or FD6 Monomer, and Single Dose Study ofFD6-IgG4 Administered by Intravenous Infusion to Cynomolgus Monkeys Xx

The purpose of this study was to evaluate the potential toxicity andtoxicokinetics of Hu5F9-G4 when administered to female cynomolgusmonkeys via a 1-hour IV infusion at escalating doses over a 5 weekperiod (FD6-IgG2 was another antibody candidate evaluated in thisstudy). In this study, one monkey was administered EPO (17,000 U/kg) onDay-5 followed by escalating doses of Hu5F9-G4 up to 300 mg/kg onceweekly on Days 1, 8, 15, 24, and 31; the other monkey was administeredescalating doses of Hu5F9-G4 (without pretreatment with EPO) up to 100mg/kg once weekly on Days 1, 8, 15, 24, and 31. Standard safetyparameters were incorporated in this study and both animals wereterminated on Day 43 (13 days after the last Hu5F9-G4 dose). The studydesign is presented in Table 4.

TABLE 4 Escalating Doses of Hu5F9-G4 Administered to Cynomolgus MonkeysNo. Female Hu5F9-G4 Dose Group Monkeys (mg/kg) Dose Days 1 3 1 (EPOpre-treatment) 10 8 (17,000 U/mg administered 30 15 on Day −5) 100 24300 31 2 1 1 (No EPO) 3 8 10 15 30 24 100 31

Both animals survived to the scheduled end of the study, and notreatment-related changes were noted in clinical signs, body weights,food consumption, coagulation and urinalysis parameters, clinicalchemistry parameters indicative of renal, hepatic, or cardiac effect,organ weights, or macroscopic or microscopic examinations.Treatment-related findings were limited to changes in hematology andclinical chemistry parameters. Consistent with previous studies,hematology changes included decreased RBC mass (RBC count, hemoglobin,hematocrit), and increased reticulocyte counts. Other changes includedincreases in MCHC and RDW and decreases in MCV were also observed.Decreases in RBC count and hemoglobin returned to near prestudy valuesfor both animals by the end of the study (Table 5). A robustreticulocyte count was observed in both animals starting at Day 3, whichreturned to near prestudy values in both animals by Day 43.

TABLE 5 Changes in Hematology Parameters Group/Animal RBC HGB RETIC No.Day (10∧6/μl) (g/dL) (10∧5/μl) 1/1501 (EPO Prestudy 5.48 13.0 0.66Pretreatment)  3 4.39 10.7 6.8  5 4.43 10.7 5.32 13 4.28 10.8 3.0 204.25 10.8 3.61 30 4.64 11.6 2.32 43 4.98 12.3 1.54 2/2501 (No Prestudy5.3 12.1 0.53 EPO)  3 4.19 9.6 3.27  5 4.47 10.3 5.86 13 4.6 11.5 5.9320 4.76 11.4 4.43 30 4.76 12.7 2.77 43 5.74 13.2 0.80

Changes in RBC morphology were consistent with previous studies andincluded minimal to marked microcytes, anisocytosis, polychromasia, andspherocytosis. As expected (based on the pharmacological action of EPO)these changes were more pronounced in Animal No. 2501 (no EPOpretreatment) compared to Animal No. 1501. Changes in RBC morphologyshowed partial or complete recovery by Day 37 (Table 6).

TABLE 6 Red Blood Cell Morphologies Group/ RBC Morphology and SeverityAnimal Study Micro- No. Day Anisocytosis cytes Polychromasia Spherocytes1/1501 3 1+ — 2+ 2+ (EPO 5 1+ — 2+ 1+ Pret- reatment) 2/2501 3 1+ — 1+3+ (No 5 1+ — 2+ 1+ EPO) 10 1+ 3+ 2+ 4+ 13 3+ 2+ 1+ 2+ 17 1+ — 2+ 1+ 203+ 2+ 1+ — 37 1+ — — — 1+ = minimal; 2+ = mild; 3+ = moderate; 4+ =marked; — = not applicable

In addition, increased lymphocyte, and monocyte counts were observed inboth animals, which were highest around Day 20 and ranged from2.58-3.7-fold above prestudy values for lymphocytes and 4.4-6.13-foldabove prestudy values for monocyte counts. Clinical chemistry changesincluded decreased haptoglobin observed in both animals, which returnedto prestudy levels by the end of the study. Increased bilirubin was alsoobserved on Day 13 for Animal No. 2501 (no EPO pretreatment).

Toxicokinetics confirmed exposure to Hu5F9-G4 in both animals, andcirculating concentrations of Hu5F9-G4 generally increased as the doseincreased. Administration of Hu5F9-G4 at doses up to 100 mg/kg resultedin a median half-life of 14 hours.

In summary, administration of escalating doses of Hu5F9-G4 administeredby a 1-hour IV infusion once weekly at doses up to 100 mg/kg (no EPOpretreatment) or 300 mg/kg (EPO pretreatment) was generallywell-tolerated by cynomolgus monkeys. Treatment-related changes werelimited to hematology (including RBC morphology) and clinical chemistryparameters, which were partially or completely reversible by the end ofthe study.

A Single or Escalating Dose Study of Hu5F9-G4 Administered by 1-HourIntravenous Infusion to Female Cynomolgus Monkeys

Based on the previous study, initial administration of Hu5F9-G4 at lowerdoses enables subsequent administration of higher doses that aretolerated in cynomolgus monkeys. The purpose of this study was toevaluate the potential toxicity and toxicokinetics of Hu5F9-G4 whenadministered as a priming dose at a low dose level followed by multiplemaintenance doses at higher dose levels. In addition, this study wasdesigned to model the potential clinical dosing schedule using apriming/maintenance-dosing regimen. In this study, female cynomolgusmonkeys were administered phosphate buffered saline (PBS) or escalatingdoses of Hu5F9-G4 (ranging from 0.1 to 30 mg/kg) by a 1-hour IV infusionas a single priming dose on Day 1 (Groups A and H). Animals in GroupsB-F were administered PBS or Hu5F9-G4 as a priming dose on Day 1,followed by multiple maintenance doses of PBS or various dose levels ofHu5F9-G4. One animal in Group D (10501) and Group F (12501) wasadministered a second priming/maintenance dose cycle starting on Day 68(priming dose of 3 mg/kg) followed by twice weekly maintenance doses (30mg/kg) for 2 weeks on Days 75, 78, 82, and 85. While the first primingdose on Day 1 for Animal No. 10501 was 1 mg/kg, the second priming doseon Day 68 was increased to 3 mg/kg to evaluate if this increase inpriming dose level would be tolerated (the priming dose on Days 1 and 68for Animal No. 12501 was 3 mg/kg). Rather than having apriming/maintenance dose schedule, the animal in Group G wasadministered Hu5F9-G4 once weekly at 10 mg/kg on Days 1, 8, 15, and 22(due to low RBC mass, the Day 15 dose was not administered). The studydesign is presented in Table 7.

TABLE 7 Administration of Hu5F9-G4 as a Priming/Maintenance DoseSchedule to Cynomolgus Monkeys Dose No. of Test Level Animal No. GroupAnimals Material Dose Day(s)^(A) (mg/kg) 1501 A 1 PBS  1 0 2501 A 1Hu5F9-G4  1 0.1 3501 A 1 Hu5F9-G4  1 0.3 4501 A 1 Hu5F9-G4  1 1 5501 A 1Hu5F9-G4  1 3 6501 A 1 Hu5F9-G4  1 10 7501 A 1 Hu5F9-G4  1 30 8501 B 1PBS  1 0  8 15 22 29 36 43 68 75 78 82 85 9501 9502 C 2 Hu5F9-G4  1 1  810 15 22 29 10 36 10 43 10 10501^(D) D 2 Hu5F9-G4  1 1 10502  8 30 15 2229 30 36 30 43 30   68^(B) 3   75^(B) 30   78^(B) 30   82^(B) 30  85^(B) 30 11501 11502 E 2 Hu5F9-G4  1 3  8 10 15 22 29 10 36 10 43 1012501^(D) 12502 F 2 Hu5F9-G4  1 3  8 30 15 22 29 30 36 30 43 30   68^(B)3   75^(B) 30   78^(B) 30   82^(B) 30   85^(B) 30 13501 G 1 Hu5F9-G4  110  8   15^(C) 22 14401 H 1 Hu5F9-G4  1 30 ^(A)Priming dose administeredon Day 1; maintenance doses administered on all subsequent days.^(B)Dosing on Days 68 (priming dose) followed by maintenance doses onDays 75, 78, 82, and 85 applicable to only 1 animal in this group(Animal 10501 in Group D and Animal 12501 in Group F). ^(C)Animal had adose holiday on this day due to low RBC mass ^(D)Animal Nos. 10501 and12501 were terminated on Day 120 and subjected to full necropsy; allother animals were returned to the Facility's animal colony on Day 120.

All animals were evaluated for changes in clinical signs, foodconsumption, body weights, clinical pathology parameters (hematology,coagulation, clinical chemistry, urinalysis). Blood samples werecollected throughout the study for toxicokinetics and evaluation of ADAresponses. Due to the level of decreased RBC mass, a dose holidayoccurred on Day 15 for the animal in Group G (13501; priming/maintenancedose of 10 mg/kg); this animal was administered the last scheduled doseon Day 22. Animals were returned to the Testing Facility colony on Day120, except for Animal Nos. 10501 (Group D) and 12501 (Group F) thatwere euthanized on Day 120, and subjected to a full necropsyexamination; organ weights and microscopic examination of tissues werealso performed.

No unscheduled deaths occurred, and overall administration of Hu5F9-G4was clinically well tolerated. Findings considered related to Hu5F9-G4were limited to changes in hematology and clinical chemistry parameters.

Single-Dose Groups (A and H): Hematology Parameters

Hematology changes were noted in animals administered a single dose ofHu5F9-G4 (Groups A and H). Variable decreases in RBC mass were observedin animals in Group A (administered escalating priming doses rangingfrom 0.1 to 30 mg/kg) during Days 3 to 14 in dose groups ≥0.3 mg/kg (nochanges were observed for 0.1 mg/kg). The decreases in RBC mass rangedup to 0.73-fold below prestudy levels for 0.3 mg/kg, 0.63-fold belowprestudy for 1 and 10 mg/kg, and 0.53-fold below prestudy for 30 mg/kg(Table 8). Interestingly, the RBC mass for the animal in Group Hdecreased only slightly below prestudy levels even though this animalwas administered the highest priming dose (30 mg/kg). These decreases,however, showed a continued trend to recovery to the last time pointevaluated for Groups A and H. Reticulocyte counts increased for allanimals (including control), indicating responsive erythropoiesis. Inaddition, increases in MCHC ≥0.3 mg/kg) and RDW(≥0.1 mg/kg) were noted,with increases up to 1.2-fold above prestudy values for MCHC and 2-foldabove prestudy values for RDW. Decreases in MCV were also noted at doses≥0.3 mg/kg, which ranged up to 0.91-fold below prestudy values. Freeplasma hemoglobin was not detected in any animal. Lymphocyte countsincreased at doses ≥0.3 mg/kg, with values ranging from 1.19- to1.86-fold above prestudy values; increases in lymphocytes correspondedwith white blood cell counts, which ranged up to 2.5-fold aboveprestudy. Changes in RBC morphologies were evaluated on Days 6 and 10,and noted at doses ≥0.1 mg/kg; these changes increased in severity withhigher doses (0.3 to 30 mg/kg) and included minimal to markedmacrocytes, microcytes, anisocytosis, polychromasia, and spherocytosis.

TABLE 8 Changes in Hematology Parameters for Animals Administered aSingle Priming Dose of Hu5F9-G4 (Groups A and H) Animal No. RBC HGB(dose of Hu5F9-G4) Study Day (10 ^(∧) 6/μl) (g/dL) Group A  1501(control) Pre-study 5.87 13.5  3 5.46 12.6  6 5.20 12 14 5.63 13 42 5.5513.1  2501 (0.1 mg/kg) Pre-study 5.62 13.9  3 5.07 12.5  6 4.67 11.6 145.27 13.1 42 5.82 14.4  3501 (0.3 mg/kg) Pre-study 5.58 12.8  3 4.9211.2  6 4.44 10.4 14 5.2 12.1 42 5.88 13.5  4501 (1 mg/kg) Pre-study5.31 13.8  3 3.75 9.7  6 3.65 9.6 14 4.39 11.5 42 5.51 14.3  5501 (3mg/kg) Pre-study 5.64 13  3 4.68 11  6 4.05 9.9 14 4.44 11.6 42 5.5613.8  6501 (10 mg/kg) Pre-study 6.24 14.2  3 5.01 11.2  6 4.14 9.7 145.24 12.1 42 6.25 14.4  7501 (30 mg/kg) Pre-study 5.22 12.5  3 4.69 11.2 6 3.02 7.3 14 3.98 9.9 42 5.37 12.8 Group H 14501 (30 mg/kg) Pre-study5.99 13.9  3 4.9 11.2  6 4.76 11.3 14 4.87 11.8 42 5.77 14Priming/Maintenance Groups (B-F) and Once Weekly Dosing (Group G):Hematology Parameters

Hematology changes observed in animals administered a priming dose onDay 1 followed by repeat maintenance doses of Hu5F9-G4 (Groups B-F) andHuF59-G4 once weekly (Group G) were consistent with those observed insingle-dose animals (Groups A and H). Variable decreases in RBC mass(RBC counts, hemoglobin, hematocrit) were observed in Groupsadministered priming doses 1 mg/kg and maintenance doses 10 mg/kg(Tables 9-10). These decreases in RBC mass tended to be greater atearlier time points (Days 5, 8 12). In addition, the decreases in RBCmass were greater for the animal administered 10 mg/kg once weekly(Animal No. 13501; Group G) compared to the other animals administeredthe priming/maintenance dose schedule. Interestingly, while thehemoglobin levels for Animal No. 10501 (Group D) decreased following thefirst priming dose of 1 mg/kg on Day 1, hemoglobin levels did not dropfollowing the second higher priming dose on Day 68 or after the secondcycle of maintenance doses on Days 75, 78, 82, and 85 (Tables 9-10).Additionally, the hemoglobin level for the other animal (No. 12501;Group F) administered a second priming/maintenance cycle remained atprestudy levels during the second cycle. These data indicate that theanemia produced by priming doses below 10 mg/kg is less severe comparedto priming doses mg/kg, and the lower priming doses allow continuedmaintenance dosing of Hu5F9-G4 that is tolerated by cynomolgus monkeys.Further, these data show that the priming/maintenance-dosing scheduledoes not produce an anemia as severe as that observed with the onceweekly dose schedule (Group G). For all priming/maintenance groups,there was a trend to recovery in RBC mass to the end of the study.Animal 13501 (Group G, ≥10 mg/kg once weekly) had a dose holiday on Day15 due to low RBC mass (hemoglobin level was 6.5 g/dL on Day 12).Hemoglobin levels, however, began to recover on Day 19, and thus, dosingresumed for this animal, which was administered the last dose on Day 22(Tables 9-10). The hemoglobin levels for Animal No. 13501 steadilymaintained a trend towards recovery, and by the last time point (Day71), returned to slightly above prestudy levels. Reticulocyte countswere increased for all Groups (B-F, including the control group), whichindicate responsive erythropoiesis. The MCHC and RDW variably increasedat priming/maintenance doses ≥1/10 mg/kg with values ranging up to1.21-fold above prestudy for MCHC and 2.41-fold above prestudy for RDW.Variable decreases (up to 0.90-fold above prestudy) in MCV were alsoobserved. Similar to the changes in RBC mass, the changes observed inMCHC, RDW, and MCV were more pronounced in Animal No. 13501 (Group G)administered 10 mg/kg once weekly compared to the other animalsadministered the priming/maintenance dose schedule. Changes in MCHC,RDW, and MCV showed a continued trend for recovery to or near prestudylevels, indicating these changes were reversible. Importantly, freeplasma hemoglobin was not observed in any animal. Changes in RBCmorphology (consistent with changes in hematology parameters) were alsoobserved, and included anisocytosis, macrocytes, microcytes (notobserved at priming/maintenance dose of 3/30 mg/kg), polychromasia, andspherocytes. Overall, the incidence and severity of these changes didnot occur in a dose-dependent manner, and based on the time pointsevaluated, showed a trend to partial or complete recovery by the end ofthe study. Lymphocyte counts also increased at priming/maintenance doses≥1/10 mg/kg, ranging from 1.25- to 2.01-fold above prestudy values, andconsistent with other parameters, overall showed a trend for recovery.

TABLE 9 Changes in Hematology Parameters for Animals AdministeredPriming/Maintenance Dosing (Groups B-F) Animal No. (priming/ RBC HGBRETIC maintenance dose) Study Day (10 ^(∧) ⁶/μl) (g/dL) (10 ^(∧) 9/L)Group B  8501 (control) Pre-study 6.01 14.1 102.2  5 5.31 12.7 98.7 125.55 13.1 169 22 5.35 12.6 228.8 43 5.54 13 181.5 64 5.65 13.5 155.1Group C  9502 (1/10 mg/kg) Pre-study 5.73 13.3 51.8  5 4.08 9.8 337.7 123.18 8.4 438.2 22 3.88 10.5 602.9 43 5.05 12.3 92.6 64 5.4 13 54.8 GroupD 10501 (1 and 3/30 mg/kg) Pre-study 5.53 13.5 42.2  5 3.91 9.9 239 123.18 8.8 479.1 22 4.11 11.5 395.5 42 4.39 11.7 317.4 64 4.86 12.8 143.368 5.03 13.2 158.9 78 4.61 12.2 290.7 88 4.37 12 362.8 106  4.62 12.1336.5 10502 (1/30 mg/kg) Pre-study 5.95 13.6 44.1  5 4.44 10 195.2 124.09 9.3 246.3 22 3.86 9.7 683.2 42 5.12 11.9 169.8 64 5.6 13 122.5Group E 11501 (1/10 mg/kg) Pre-study 5.36 12 50.9  5 3.8 8.5 171.9 123.84 8.8 381.8 22 4.69 10.8 314.5 42 5.17 11.2 115.3 64 5.37 11.9 56.211502 (1/10 mg/kg) Pre-study 5.85 14.2 94.4  5 4.42 10.8 234 12 4.2110.7 812.8 22 4.77 11.8 497.6 42 5.02 11.8 157.7 64 5.84 14.2 127.3Group F 12501 (3 and 3/30 mg/kg) Pre-study 5.54 13 61.9  5 4.46 10.7202.6 12 4.32 10.8 330 22 4.97 11.6 212.3 42 4.72 11.5 151.5 64 5.3312.8 135 68 5.75 13.5 112 78 5.3 12.9 134.3 88 5.03 12.3 164.1 106  5.413.1 174.1 12502 (3/30 mg/kg) Pre-study 5.26 12.6 62.9  5 4.09 10 181.312 4.07 10.1 222.2 22 4.53 11.1 275.7 42 4.75 11.2 196.2 64 5.04 12.6115

TABLE 10 Changes in Hematology Parameters for Animal No. 13501 (Group G)Administered 10 mg/kg Hu5F9-G4 Once Weekly Group/Animal Study RBC HGBRETIC No. Day (10 ^(∧) ⁶/μl) (g/dL) (10 ^(∧) 9/L) Group Pre-studyG/13501^(A) 5 3.70 8.6 217.6 12 2.73 6.5 196.3 19 3.63 9.1 312.7 22 4.1910.3 230.1 40 5.48 13.1 101.3 50 5.37 12.8 82.1 71 6.01 13.8 75.8 ^(A)Adose holiday occurred on Day 15

Single-Dose Groups (A and H): Clinical Chemistry Parameters

In single dose animals (Groups A and H), increase in total bilirubin wasnoted at doses ≥1 mg/kg, with the increases occurring on Day 6 andranged from 1.56- to 3.29-fold above prestudy values; increases in totalbilirubin did not occur in a dose-dependent manner and showed a trend torecovery. An increase in ALT and AST was noted in a single animaladministered 30 mg/kg (Animal 14501; Group H) on Day 6, but theseincreases showed a trend to recovery by the last time point. Haptoglobindecreased at doses ≥0.1 mg/kg, however, haptoglobin was below detectionlevels at 3 to 4 time points for all animals, including prestudy for twoHu5F9-G4-treated animals and one PBS control animal on Day 42. Thus, thedecreases in haptoglobin were considered to have an uncertainrelationship to single dose administration of Hu5F9-G4 in this study.

Priming/Maintenance Groups (B-F) and Once Weekly Dosing (Group G):Clinical Chemistry Parameters

Similar to the single dose groups, total bilirubin was increased in allgroups administered the priming/maintenance dose schedule; levels intotal bilirubin, however, remained below 1 mg/dL throughout the study(Table 11). Increases in total bilirubin showed a trend to recover atthe end of the study. Haptoglobin levels decreased for all groups, whichshowed a trend to recover at the end of the study (Table 11). Sporadicchanges in ALT, AST, and LDH occurred in a couple of animals at sometime points during the course of the study; however, these changes, werewithin normal limits (based on the Test Facility's historical database),occurred on only 1 or 2 days during the study period, and were transientin nature.

TABLE 11 Priming/Maintenance (Groups B-F) and Once Weekly Dosing (GroupG): Clinical Chemistry Parameters Total Bili HAPTO Group/Animal No.(dose) Study Day (mg/dL) (g/L) Group B  8501 (control) Pre-study 0.230.48  5 0.31 0.46 12 0.23 0.92 26 0.22 0.7 40 0.21 0.66 78 0.13 0.61Group C  9502 (1/10 mg/kg) Pre-study 0.17 0.3  5 0.28 0.15 12 0.72 0.1526 0.2 0.15 57 0.18 0.34 Group D 10501 (1 and 3/30 mg/kg) Pre-study 0.280.4  5 0.25 0.15 12 0.53 0.15 26 0.35 0.15 47 0.32 0.15 68 0.21 0.15106  0.38 0.15 10502 (1/30 mg/kg) Pre-study 0.22 0.95  5 0.22 0.94 120.34 0.15 26 0.21 0.15 57 0.16 0.41  5 0.22 0.94 Group E 11501 (1/10mg/kg) Pre-study 0.23 0.36  5 0.28 0.34 26 0.22 0.55 40 0.17 0.69 570.22 0.41 11502 (1/10 mg/kg) Pre-study 0.12 0.61  5 0.18 0.15 12 0.330.15 26 0.2 0.44 40 0.13 1.81 57 0.12 0.6 Group F 12501 (3 and 3/30mg/kg) Pre-study 0.39 0.35  5 0.38 0.3 12 0.52 0.15 26 0.27 0.15 47 0.250.33 68 0.25 0.15 106  0.34 0.15 12502 (3/30 mg/kg) Pre-study 0.2 0.32 5 0.39 0.31 12 0.54 0.34 26 0.37 0.15 40 0.29 0.34 57 0.25 0.15 Group G13501 (10 mg/kg once weekly) Pre-study 0.15 0.9  5 0.56 0.15 26 0.190.61 40 0.12 0.73 71 0.12 0.79

Animal Nos. 10501 (Group D) and 12501 (Group F) were euthanized on Day120, and subjected to a full necropsy examination; organ weights andmicroscopic examination of tissues were also performed. While a single,minimal focus of white matter degeneration characterized by axonaldegeneration with phagocytes and mononuclear cell infiltrates was notedwithin the medulla oblongata of Animal No. 10501, it is unknown if thisfinding was incidental or related to Hu5F9-G4 due to the minimal natureof the finding and the small number (2) of animals subjected tomicroscopic examination. Importantly, this finding was not noted in thepivotal GLP 8-week toxicology study, which indicates that this findingwas likely incidental in nature.

Toxicokinetics showed that no measurable concentrations of Hu5F9-G4 wereobtained for Groups administered a single dose of Hu5F9-G4 at doses ≤0.3mg/kg. C_(max) and AUC_(0-t) generally increased as the dose increased,and increases in C_(max) appeared to be greater than dose proportionalfor doses ≥1 mg/kg. Increases in AUC_(0-t) were also not proportionalwith dose. The T_(1/2) for the 10 and 30 mg/kg dose levels were 10.7 to46.5 hours, respectively, and suggests T_(1/2) may increase with anincrease in dose.

For groups administered repeat doses, following a priming dose of 1 or 3mg/kg (on Day 1 or 68) and repeat administration of 10 or 30 mg/kg, meanC_(max) increased from Day 1 to Day 8; this increase in C_(max) wasgreater than dose proportional from Day 1 to Day 8. Following a primingdose of 1 (Day 1) or 3 (Day 68) mg/kg and maintenance dosing of 30mg/kg, mean C_(max) increased from Day 1 to Day 8; this increase inC_(max) was greater than dose proportional from Day 1 to Day 8.Half-life ranged from 10.8 to 173 hours. Some animals had lower thanexpected serum concentrations of Hu5F9-G4, which suggests the presenceof ADA.

In summary, administration of Hu5F9-G4 as a single priming dose up to 30mg/kg or as a priming/maintenance dose of up to 3/30 mg/kg for up to 2priming/maintenance dose schedules was clinically well tolerated incynomolgus monkeys. While once weekly administration of Hu5F9-G4 at 10mg/kg was clinically well tolerated, a dose holiday was necessary on Day15 due to the low RBC mass; however, the hemoglobin level showed a trendfor recovery on Day 19, and thus, dosing resumed for this animal.Treatment-related changes were limited to alterations in hematology(including RBC morphology) and clinical chemistry parameters. Decreasesin RBC mass were consistent with previous studies, and the decreases inRBC count and hemoglobin are likely associated with the postulatedpharmacological action of Hu5F9-G4 by binding to CD47 on aged RBCs andaccelerating their clearance. It is believed that this clearance of agedRBCs results in the initial anemia and compensating reticulocytosisobserved at early time points to replace the aged RBCs with youngerRBCs. All of the treatment-related changes in hematology and clinicalchemistry parameters showed trends for partial or complete recovery bythe end of the study, indicating that these effects of Hu5F9-G4 arereversible. Importantly, the data demonstrate that the anemia induced bythe priming/maintenance dose schedule is not as severe as with onceweekly dosing, and priming doses ≤10 mg/kg are better tolerated. Assuch, the priming/dose schedule, with a priming dose ≤10 mg/kg was usedin subsequent studies.

Pharmacokinetics of Hu5F9-G4 Following Intravenous InfusionAdministration to Cynomolgus Monkeys

The purpose of this study was to evaluate the potential toxicity andtoxicokinetics of Hu5F9-G4 when administered by a 1-hour IV infusion asa priming/maintenance dose schedule at dose levels not evaluated inprevious studies. In this study, Hu5F9-G4 was administered to malecynomolgus monkeys as a priming dose at 5 mg/kg on Day 1, followed bytwice weekly maintenance doses at 150 mg/kg on Days 8, 11, 15, 18, 22,and 25 The study design is presented in Table 12.

TABLE 12 Priming/Maintenance Priming and Maintenance Dosing of Hu5F9-G4in Cynomolgus Monkeys Hu5F9-G4 Hu5F9-G4 Priming Dose Day MaintenanceDose Days No. Male Dose Priming Dose Maintenance Group Monkeys (mg/kg)Dose (mg/kg) Dose 1 2 5 1 150 8, 11, 15, 18, 22, 25

The animals were evaluated for changes in clinical signs, body weight,hematology, coagulation, and clinical chemistry parameters (collected upto Day 77); blood samples were also collected to evaluate receptoroccupancy using flow cytometry, however, the data is not presented inthis IND. Samples were collected throughout the study up to Day 149 fortoxicokinetics and assessment of ADA responses. Both animals werereturned to the Test Facility animal colony at the end of the study.

Both animals survived to the scheduled end of the study, and no evidenceof treatment-related effects was noted in clinical signs, foodconsumption, or body weight. Treatment-related findings were observed inboth animals and included changes in hematology and clinical chemistryparameters. Consistent with previous studies, mild anemia was noted inboth animals on Day 5 (4 days after the 5 mg/kg priming dose). Asignificant reduction in RBC count was observed in both animalsbeginning on Day 8 (Animal 1036) and Day 11 (Animal 1037). On Day 18, atrend for the RBC returning to normal levels was observed in Animal1036; however, Animal 1037 continued to show significant reductions inRBC count (Table 13). The reduction in RBC count coincided with asignificant increase in reticulocytes for both animals (Table 13).Similar to the RBC count, a trend for reticulocytes returning to normallevels was observed for Animal 1036; however, reticulocytes continued toincrease for Animal 1037. In addition, hemoglobin levels weresignificantly reduced in Animal 1037 (starting on Day 15). Thehemoglobin levels for Animal 1036 also decreased, however, the decreaseobserved in this animal was not as significant as Animal 1037, andremained above 10.0 g/dL during the study except on Day 11 where thehemoglobin dropped slightly below 10.0 g/dL (Table 13). Free plasmahemoglobin was not observed in either animal. Dosing was stopped on Day15 for Animal 1037 due to the severe anemia. Dosing was not resumed forthis animal to assess if the anemia would recover; thus, Animal No. 1037was not dosed on Days 18, 22, and 25. Since the hemoglobin levels forAnimal 1036 remained above 10.0 g/dL throughout the majority of thestudy, dosing continued as planned. Despite the anemia, no clinicalsigns indicative of toxicity were observed in either animal. Inaddition, no major changes in other clinical pathology parameters wereobserved, including white blood cell counts, platelets, or creatininelevels. Both animals were examined by a staff veterinarian on Day 22with special attention to palpation of the spleen; palpation of thespleen revealed no abnormalities in either animal.

TABLE 13 Changes in Hematology Parameters Animal RBC HGB RETIC No. StudyDay (10 ^(∧) ⁶/μl) (g/dL) (10 ^(∧) 9/L) 1036 Prestudy 5.87 13.8 75.4 54.24 10.4 234.6 8 3.92* 10 984.3 11 3.75* 9.8 847.7 15 3.94* 10.3 557.518 4.3 10.8 542.1 22 4.45 11.6 414.5 25 4.51 11.5 329.4 29 4.59 11.7374.2 48 5.07 12.9 236.1 77 5.25 13.5 258.6 1037 Prestudy 6.47 14.3 51.45 4.85 11.1 232 8 4.13 10 937.1 11 3.24* 8 612.9 15 2.23* 5.5* 670.3 181.86* 4.7* 749.7 22 1.95* 5.1* 1076 25 2.02* 5.7* 1043 29 2.32* 6.6*1085 48 4.71 11.9 1106 77 6.31 14.9 505.7 *Value substantially outsideof the normal range for this parameter in the cynomolgus monkey based onthe Testing Facility's historical database

The RBC and hemoglobin levels in Animal 1037 remained significantlyreduced and reticulocytes remained significantly elevated (compared toprestudy) up to Day 29. However, by Day 48, the RBC, hemoglobin, andreticulocytes began to recover, and by Day 77, these parameters returnedto prestudy levels. Additionally, the changes in these hematologyparameters observed in Animal 1036 began to recover on Day 22, andreturned to prestudy levels by Day 77. Thus, while Animal 1037 appearedto be more sensitive to the anemia associated with the administration ofHu5F9-G4, termination of dosing (on Day 15) shows that over time, theanemia is reversible. Changes in RBC morphology were also observed inboth animals, and consistent with the previous studies, included minimalto marked anisocytosis, microcytes, polychromasia, and spherocytes.

Toxicokinetics showed that serum Hu5F9-G4 concentrations were similarbetween the two animals from Day 1 through 4 hours post-dose on Day 15(the dose holiday for Animal 1037 began on Day 15). The T_(1/2) for bothmonkeys ranged from 173 to 212 hours.

In summary, the treatment related effects in this study was consistentwith the previous studies, and included changes in hematology andclinical chemistry parameters. All of these treatment-related changes,including the severe anemia observed in Animal 1037, were reversible,and returned to normal ranges by the end of the study. In addition,despite the anemia observed, no clinical signs of toxicity were observedin either animal.

8-Week Toxicity Study of Hu5F9-G4 by Intravenous Infusion in CynomolgusMonkeys with an 8-Week Recovery Period

The purpose of this GLP study was to evaluate the potential toxicity andtoxicokinetics of Hu5F9-G4 when administered to cynomolgus monkeys as apriming dose followed by repeat maintenance doses. Due to the severeanemia with the priming/maintenance dose of 5/150 mg/kg observed inAnimal No.1037 in the previous study, the priming and highestmaintenance dose used in this study was 5/100 mg/kg to provide areasonable safety margin for the doses proposed in the clinical study.Vehicle or Hu5F9-G4 (5 mg/kg) was administered via a 1-hour IV infusionas a priming dose at 5 mg/kg on Day 1 followed by maintenance doses ofvehicle or Hu5F9-G4 at doses of 5, 10, 50, or 100 mg/kg administeredtwice weekly for 7 consecutive weeks (Days 8, 11, 15, 18, 22, 25, 29,32, 36, 39, 43, 46, 50, and 53). The same dose level (5 mg/kg) wasemployed for the priming and maintenance doses for Group 5. Recoveryanimals were included in the vehicle and high dose groups to assess thereversibility of any treatment-related effect. The study design ispresented in Table 14.

TABLE 14 8-Week Toxicology Study in Cynomolgus Monkeys Dose^(A) (mg/kg)No. Males/ No. Males/ Group Priming Maintenance Females Females 1 0 0(vehicle) 3/3 2/2 (vehicle) 2 5 10 3/3 — 3 5 50 3/3 2/2 4 5 100 3/3 2/25 5 5 2/2 ^(A)The priming dose was administered on Day 1, and themaintenance dose was administered twice weekly on Days 8, 11, 15, 18,22, 25, 29, 32, 36, 39, 43, 46, 50, and 53.

Safety pharmacology parameters were incorporated into this study andincluded assessment of respiratory function (visual respiratory rate,cardiovascular function (ECGs), and changes in clinical signs (e.g.,activity, behavior) indicative of an effect on central nervous systemfunction. Blood samples were collected throughout the study fortoxicokinetics and assessment of ADA responses, as well as to evaluatereceptor occupancy of CD47. Throughout the duration of the study,clinical pathology samples were evaluated to assess specific parameters(e.g., hemoglobin, RBC, reticulocytes) based on data from previousstudies. Severe anemia was observed in two animals (Animal No. 3002;Group 3, priming/maintenance dose 5/50 mg/kg; Animal No. 4504; Group 4,priming/maintenance dose 5/100 mg/kg), and these animals were placed ondose holidays to evaluate the recovery of the anemia and how the animalsresponded once dosing resumed. Animal No. 3002 had dose holidays formaintenance doses 6-9 (Days 25, 29, 32, and 36), and dosing resumed onDay 39 (maintenance dose 10). Animal No. 4504 was place on dose holidayon Day 25 (dose 6), and continued on dose holiday until the end of thedose period. Main study animals were terminated on Day 57 and recoveryanimals were terminated on Day 109. The in-life portion of the study iscomplete, and all data for main study animals, including histopathology,is available. Data from recovery animals will be submitted whenavailable.

No unscheduled deaths occurred in this study, and administration ofHu5F9-G4 was clinically well-tolerated. No treatment-related effectswere observed in clinical signs, body weights, physical andophthalmology examinations, body temperature, ECGs, respiration or heartrate, coagulation or urinalysis parameters, organ weights, ormacroscopic and microscopic examinations.

Consistent with the previous pilot studies, treatment-related changeswere observed in all Hu5F9-G4-treated groups in hematology parametersand included mild to moderate decreases in red cell mass (including RBCcount, hemoglobin, and hematocrit), which were most pronounced followingthe priming dose on Day 1; these changes in hematology parameters showeda continued trend to recover by the end of the dosing phase (see Table15 for changes in hemoglobin levels). While the decrease in RBC count,hemoglobin, and hematocrit were observed in all Hu5F9-G4-treated groups,these changes did not occur in a clear dose-dependent manner. Increasesin reticulocytes were observed in all Hu5F9-G4-treated groups,indicative of a robust erythropoietin response associated with thedecreases in RBC mass. Consistent with the previous studies, thedecreased red cell mass was associated with decreases in MCV andhaptoglobin, and increases in MCHC, reticulocytes, and RDW. Free plasmahemoglobin was not observed in any dose groups. Minimal to mildincreases in lymphocytes were also observed, but these increases weretransient and sporadic in nature and did not occur in a dose-dependentmanner. A minimal to mild increase in platelets was observed on Day 8(which was statistically significant compared to control for mostgroups), but began to return to control values in most groups by Day 11.This increase in platelets was considered to be a reactivethrombopoiesis and physiological response to the acceleratederythropoiesis, which was evident by concomitant increases inreticulocytes. All of these treatment-related changes in hematologyparameters were partially or completely reversible by the end of thedosing phase.

TABLE 15 Changes in Hemoglobin Levels Animal No. Study Day HGB (g/dL)Group 1 (vehicle control) 1001 Prestudy 13.3  3 12.3  8 13.8 11 12 1512.5 25 12.5 39 13.2 57 12.4 1002 Prestudy 13.8  3 12.8  8 13.6 11 12.515 12.9 25 12.6 39 13.1 57 12.6 1003 Prestudy 13.8  3 13.1  8 13.5 1112.7 15 13 25 13.4 39 13.2 57 13 1104 Prestudy 14.6  3 14.8  8 14.9 1113.7 15 13.8 25 14.1 39 13.8 57 12.4 74 13.8 109 14.7 1005 Prestudy 14.8 3 13  8 13.9 11 11.9 15 12.4 25 13 39 13.1 57 12 74 12.6 109  13.6 1501Prestudy 12.9  3 11.7  8 12.3 11 11.4 15 11.6 25 11.9 39 11.9 57 11.51502 Prestudy 13  3 11.9  8 12.5 11 11.7 15 11.8 25 12.4 39 12.2 57 13.41603 Prestudy 13.7  3 12.3  8 12.7 11 11.2 15 12.4 25 12.3 39 11.5 5710.6 1504 Prestudy 15.1  3 13.5  8 13.8 11 13 15 12.9 25 12.6 39 13.1 5712.7 74 13.1 109  13.8 1505 Prestudy 13.6  3 12.5  8 13.3 11 13.1 1512.6 25 12.8 39 12.9 57 9.9 74 12.6 109  13.1 Group 2 (5/10 mg/kg) 2001Prestudy 14.1  3 12.6  8 12.5 11 11.4 15 12.4 25 12.6 39 12.7 57 12 2002Prestudy 15.9  3 12.1  8 11.8 11 10.8 15 11.6 25 13.5 39 13.8 57 12.62003 Prestudy 14.1  3 11.5  8 11.2 11 9.8 15 10.1 25 11.9 39 12.7 5711.6 2501 Prestudy 13.7  3 11.3  8 10.7 11 8.8 15 8.9 25 11.4 39 13.0 5711.7 2502 Prestudy 13  3 10.4  8 10.2 11 9.1 15 10.1 25 10.5 39 11.4 579.9 2503 Prestudy 14.3  3 12  8 12.1 11 9.9 15 10 25 11.5 39 13 57 12.1Group 3 (5/50 mg/kg) 3001 Prestudy 14.9  3 12.8  8 12.1 11 11.2 15 11.725 13.2 39 14.1 57 13.2 3003 Prestudy 13.9  3 10.8  8 11.3 11 10.1 15 1025 12.4 39 13.4 57 12 3104 Prestudy 14.6  3 13  8 13.7 11 12.3 15 11.825 12.7 39 13.4 57 12.3 74 13 109  13.8 3005 Prestudy 14.6  3 12.1  8 1311 11.9 15 12.1 25 13.3 39 13.6 57 12.5 74 13.1 109  12.5 3601 Prestudy15  3 11.4  8 12 11 10.2 15 10.9 25 11.6 39 11.8 57 10.4 74 12 109  14.13602 Prestudy 13.2  3 10.9  8 10.3 11 8.7 15 8.8 25 11.2 39 12.2 50 8.157 7 67 8.8 74 10.9 109  13.4 3503 Prestudy 13  3 10.4  8 10.1 11 9 158.5 25 10.2 39 11 57 10.1 3504 Prestudy 12.8  3 10.9  8 11.9 11 10.5 1510.6 25 10.7 39 10.8 57 11 3505 Prestudy 13.4  3 11  8 11.1 11 10.2 1510.8 25 11.1 39 12 57 11.4 Group 4 (5/100 mg/kg) 4001 Prestudy 13.5  310.5  8 9.8 11 8.9 15 9.3 25 10 39 12.1 57 11.5 4102 Prestudy 13.5  310.7  8 9.5 11 9.2 15 10 25 11.4 39 11.3 57 11.4 4103 Prestudy 14.8  311.3  8 10.4 11 9.5 15 10.2 25 12 39 13.5 57 12.4 4004 Prestudy 13.8  310.9  8 10 11 9.1 15 9.8 25 12.4 39 12.8 57 11.9 74 12.4 109  12.5 4005Prestudy 14.2  3 11.6  8 11.4 11 9.6 15 8.8 25 11.1 39 12.4 57 11.1 7412.2 109  12.7 4501 Prestudy 13  3 9.7  8 10.4 11 8.4 15 8.4 25 8.8 3910.4 57 10.4 4502 Prestudy 13.5  3 11.7  8 11.8 11 9.7 15 11 25 11.9 3912.9 57 12.3 4503 Prestudy 13.6  3 11.4  8 12.1 11 11 15 10.6 25 13 3913.5 57 13.2 4505 Prestudy 13.3  3 11.5  8 11.3 11 9.8 15 9.1 25 10.2 3911.2 57 11 Group 5 (5/5 mg/kg) 5001 Prestudy 14.4  3 12.2  8 11.9 1111.7 15 12.4 25 13.1 39 14.5 57 13.3 5002 Prestudy 14.3  3 12.4  8 11.911 11.5 15 11.7 25 12.2 39 12.8 57 12.8 5501 Prestudy 13.9  3 10.7  810.7 11 9.8 15 10.1 25 11.7 39 13.6 57 12 5502 Prestudy 14  3 11.4  810.9 11 10.6 15 10.9 25 12.4 39 12.9 57 12

While the hemoglobin decreased in all Hu5F9-G4-treated animals followingthe administration of the priming dose on Day 1, the decrease inhemoglobin was generally most pronounced following administration of thefirst maintenance dose on Day 8 (see Day 11 hemoglobin levels in Table16). The extent of the decrease in hemoglobin was varied across animals,and the incidence of animals having a hemoglobin level ≤10.0 g/dL on Day11 was 67%, 30%, 90%, and 50% for Groups 2 (2/10 mg /kg), 3 (5/50mg/kg), 4 (5/100 mg/kg) and 5 (5/5 mg/kg), respectively. While theanemia (on Day 11) did not occur in a clear dose-dependent mannerbetween Groups 2, 3, or 5, Group 4 had the highest number of animalsthat had a hemoglobin level ≤10.0 g/dL. Overall, a continued trend inrecovery in hemoglobin was observed across animals, starting around Days15 to 32 and continuing until the end of the study. One exception,however, was noted near the end of the study where Animal No. 3602 hadanother substantial reduction in hemoglobin following administration ofthe 11^(th) maintenance dose Day 46 (hemoglobin decreased as low as 7.0g/dL on Days 55 and 57; Table 16). The hemoglobin level for Animal No.3602, however, began to recover on Day 60 (7 days after the lastmaintenance dose), and returned to prestudy levels (13.4 g/dL) by theend of the study on Day 109. Due to the severe anemia observed, twoanimals (Animal No. 3002; Group 3, priming/maintenance dose 5/50 mg/kg;Animal No. 4504; Group 4, priming/maintenance dose 5/100 mg/kg) wereplaced on dosing holidays to evaluate the recovery of the anemia and howthe animals respond once dosing resumed. Animal No. 3002 showed moresevere anemia (as low as 5.7 g/dL on Days 15 and 18) and had doseholidays for maintenance doses 6-9 (Days 25, 29, 32, and 36); dosingresumed on Day 39 (maintenance dose 10). Animal No. 4504 was place ondose holiday on Day 25 (dose 6), and continued on dose holiday until theend of the dose period (see Table 16 to see changes in hemoglobinlevels). The changes in RBC count, hemoglobin, and reticulocytes notedin Animal No. 3002 began to recover on Day 36, and continued to recoverto the end of the study on Day 57. Likewise, the hematology changes inAnimal No. 4504 started to recover, and showed a continued trend torecovery until the end of the study (Day 109; this animal was in therecovery group). Thus, while it appears that a low number of animals maybe especially sensitive to the anemia produced by Hu5F9-G4, the anemiais transient and the hemoglobin levels recover over time.

TABLE 16 Hematology Parameters for Animals Placed on Dose Holiday HGBGroup/Animal No. Study Day (g/dL) 3/3002^(A) Pre-study 14.4 3 10.8 8 9.611 6.9 15 *5.7 18 *5.7 25 *5.9 36 12.2 46 10.3 57 11 4/4504^(B)Pre-study 13.7 3 11.2 8 11.7 11 9.6 15 7.3 18 *6.9 25 7.1 36 7.2 46 8.457 11 74 12.9 109 13 ^(A)Animal No. 3002 was placed on dose holiday onDay 25 and dosing resumed on Day 39; main study animal and terminated onDay 57 ^(B)Animal 4504 was placed on dose holiday on Day 25 and remainedon dose holiday to the end of the study; recovery animal and terminatedon Day 109 *Significantly below the normal range for cynomolgus monkeysbased on Testing Facility's historical database

Changes in blood cell morphology were consistent with previous studiesand considered to be associated with accelerated red blood celldestruction/clearance and increased erythropoiesis. These changes rangedfrom minimal to marked in nature and included anisocytosis, spherocytes(microcytes), polychromasia, as well as eccentrocytes and atypicalerythrocyte fragments consistent with erythrocyte injury/clearance. Thevariability in the range of red blood cell size was due to the mixtureof smaller spherocytes and larger polychromatophils (reticulocytes). Atransient increase was also observed in the number of nucleatederythrocytes in the circulation of several Hu5F9-G4-treated animals.Changes in red blood cell morphology showed continued trends of recoveryto the end of the study. Changes in bone marrow smear evaluations wereminimal to moderate and limited to morphologic changes in the erythroidlineage (dysplasia), which consisted of occasional cells with abnormalnuclear shapes, multiple nuclei, nuclear blebbing, and/or nuclear tocytoplasm maturation asynchrony (abnormal nucleus to cytoplasmmaturation). Additional changes were considered to be related to theaccelerated erythropoietin response associated with Hu5F9-G4 whichincluded mild decreases in the mean M:E ratio in Group 3 and Group 4(females only) animals along with an appropriate minimal to mild shiftto more immature erythroid precursors associated with acceleratederythropoiesis.

Consistent with previous studies, treatment related changes inhematology parameters (i.e., decreased RBC and hemoglobin; increasedreticulocytes) were associated with increases in total bilirubin anddecreases in haptoglobin. Other changes in clinical chemistry parameterswere observed only in the high dose group (5/100 mg/kg) and included aslight decrease in albumin (two female animals), a slight increase inglobulin, and a corresponding decrease in albumin:globulin ratio. Alltreatment-related changes in clinical chemistry parameters werepartially or completely reversible at the end of the dosing phase.

The Day 8 toxicokinetics showed that following a priming dose of 5 mg/kgon Day 1, and an initial maintenance dose at 5, 10, 50 or 100 mg/kg onDay 8, increases in C_(max) were dose proportional from 10 to 100 mg/kg,but greater than dose proportional from 5 to 100 mg/kg. While increasesin AUC₀₋₇₂ trended towards dose proportional between 50 and 100 mg/kg,changes in AUC₀₋₇₂ were greater than dose proportional from 5 to 100 or10 to 100 mg/kg. The mean T_(1/2) appeared to increase with increasingdose and ranged from 6 hours at 5 mg/kg to 52 hours at 100 mg/kg. Therewere no obvious gender differences in exposure. Day 25 toxicokineticsshowed that following twice weekly dosing at 5, 10, 50 or 100 mg/kg for3 weeks, increases in exposure (C_(max), AUC₀₋₇₂) were greater than doseproportional from 5 to 100 mg/kg but dose proportional from 10 to 100mg/kg. In addition, exposure trended lower in female compared to malemonkeys. The apparent T_(1/2) was shorter at 5 mg/kg relative to thehigher doses. In some animals, T_(1/2) was similar between Day 25 andDay 8, and in other animals, T_(1/2) appeared to be longer on Day 25;the mean T_(1/2) ranged from 6.3 hours (5 mg/kg) to 66 hours (50 mg/kg).Day 53 toxicokinetics following twice weekly dosing at 5, 10, 50 or 100mg/kg for 7 weeks, showed increases in exposure (C_(max), AUC₀₋₇₂) weregreater than dose proportional from 5 to 100 mg/kg but dose proportionalfrom 10 to 100 mg/kg.

Concentration vs. time profiles on Day 53 relative to Day 25 or Day 8suggested that circulating concentrations of Hu5F9-G4 continued toincrease with repeat dosing. With the exception of the 5 mg/kg dose(which appeared to be impacted by ADA), mean and median exposure(C_(max), AUC₀₋₇₂) on Day 53, within each dose group, were generallyhigher than that on Day 25 or Day 8, suggesting further accumulation ofHu5F9-G4 with continued twice weekly dosing. While the development ofADA appears to impact exposure, this impact was primarily noted at the 5mg/kg maintenance dose, and overall, exposure was maintained throughoutthe study at doses ≥10 mg/kg maintenance doses.

In summary, treatment-related findings were consistent with previousstudies, and included changes in hematology and clinical chemistryparameters and bone marrow cytology. Changes in hematology parametersincluded decreases in RBC count and hemoglobin combined withreticulocytosis. Importantly, free plasma hemoglobin was not detected inany animal throughout the study. While the anemia related to Hu5F9-G4did not occur in a clear dose-dependent manner between Groups 2, 3, or5, the number of animals having a hemoglobin level 10.0 g/dL wasgreatest in Group 5, which was administered the highest maintenance dose(100 mg/kg). While the hemoglobin levels generally showed a trend inrecovery across all animals starting around Days 15-32 and continued tothe end of the study, a substantial decrease in hemoglobin was notedagain in one animal (Group 3) near the end of the study following theadministration of the 11^(th) maintenance dose on Day 46. The hemoglobinfor this animal, however, returned to prestudy levels by the end of thestudy (Day 109). Two animals (one each in Groups 3 and 4) were placed ondose holiday due to the severe anemia observed in each animal.Importantly, despite the severe anemia observed in these animals, noclinical signs of toxicity were noted and the hemoglobin level in eachanimal showed a continued trend to recovery until the study end. Changesin red blood cell morphology were consistent with accelerated red bloodcell destruction/clearance and increased erythropoiesis and consisted ofatypical erythrocyte fragments, anisocytosis, spherocytes (microcytes),and polychromasia. Changes in hematology parameters were associated withincreased total bilirubin and decreased haptoglobin. Othertreatment-related changes in clinical chemistry parameters were notedonly in the high dose group and included a slight decrease in albumin,slight increase in globulin, and the corresponding albumin:globulin(A:G) ratio. All of these treatment-related changes were eitherpartially or completely reversible in all Hu5F9-G4-treated groups by theend of the study. Changes in bone marrow cytology were limited tomorphologic changes in the erythroid lineage consisting of occasionalcells with abnormal nuclear shapes, multiple nuclei, nuclear blebbing,and/or nuclear to cytoplasm maturation asynchrony.

Overall, administration of Hu5F9-G4 via a 1-hour IV infusion as apriming dose of 5 mg/kg in Week 1 (Day 1), followed by twice weeklymaintenance doses for 7 consecutive weeks at doses up to 100 mg/kg wasclinically well-tolerated in cynomolgus monkeys. Despite thetreatment-related anemia, including the animals having dose holidays, nosigns of clinical toxicity were observed. The changes observed in thisstudy were consistent with previous studies, and considered to berelated to the pharmacological action of Hu5F9-G4 in accelerating theprocess of aging RBC elimination through binding to CD47 expressed onRBCs. Therefore, based on the totality of the data, thehighest-non-severely-toxic-dose (HTNSTD) for this study was consideredto be the priming/maintenance dose of 5/100 mg/kg, the highest doseevaluated.

Genotoxicity

The range and type of genotoxicity studies routinely conducted for smallmolecule drug products are generally not applicable tobiotechnology-derived products [ICH S6(R1)]. It is not expected that amonoclonal antibody, such as Hu5F9-G4, would interact directly with DNAor other chromosomal material. Thus, mutagenicity studies are consideredinappropriate and are not planned.

Carcinogenicity

No carcinogenicity studies have been conducted with Hu5F9-G4. Based onthe mechanism of action of Hu5F9-G4, it is not expected to becarcinogenic. Additionally, Hu5F9-G4 is neither a growth factor norimmunosuppressant. Thus, given the intended patient population and lackof mechanistic concern, carcinogenicity studies are not planned.

Reproductive and Developmental Toxicity

Reproductive and developmental toxicity studies have not been performedwith Hu5F9-G4. While a formal, stand-alone fertility study will not beperformed; no treatment-related effects were noted in the microscopicexamination of male and female reproductive organs in the 8-weektoxicity study. Since the potential teratogenic effects of Hu5F9-G4, ifany, in laboratory animals is unknown, Hu5F9-G4 should not beadministered to pregnant women. To avoid pregnancy, appropriateprecautions will be taken for male and female patients of child bearingpotential enrolled in the proposed Phase 1 clinical study (e.g., womenmust demonstrate a negative pregnancy test, patients must consent toadequate contraceptive precautions, etc.).

Local Tolerance

No stand-alone local tolerance studies have been performed; however,consistent with ICH S6(R1), evaluation of local tolerance of Hu5F9-G4(clinical observations, macro- and microscopic examination of tissuesamples from the injection site) were performed as part of the repeatdose toxicity studies.

Discussion and Conclusions

A comprehensive series of toxicology studies was conducted in support ofadministration of Hu5F9-G4 in the proposed clinical trial. These studiesincluded an in vitro hemolysis study, single- and multiple-dose studiesin rhesus and cynomolgus monkeys, and a tissue cross reactivity study ina panel of human tissues.

The major and consistent treatment related finding across all of themonkey studies was anemia (as reflected in decreased RBC count andhemoglobin). The anemia generally developed following administration ofthe first dose (or priming dose in the priming/maintenance dose schedulestudies), and was associated with changes indicative of the acceleratedclearance of RBCs and responsive erythropoiesis, includingreticulocytosis, and alterations in RBC morphologies such asanisocytosis, polychromasia, and spherocytosis. Importantly, free plasmahemoglobin was not observed across all of the studies. The changes inhematology parameters associated with Hu5F9-G4 were generally associatedwith changes in haptoglobin and total bilirubin; Haptoglobin was often,but not always reduced, and increases in total bilirubin were generallyobserved; changes in haptoglobin and total bilirubin, however, did notoccur in a dose-dependent manner. Across all studies, changes inhematology and clinical chemistry parameters associated with Hu5F9-G4showed trends of recovery during the study, and were either partially orcompletely reversible by the end of the study. Changes in bone marrowcytology (performed in the GLP 8-week study; PRO13) were considered tobe associated with accelerated erythropoiesis and included morphologicchanges in the erythroid lineage, decreases in the mean M:E ratio, andan appropriate shift to more immature erythroid precursors associatedwith accelerated erythropoiesis.

Based on the known role of CD47 in the normal clearance of aging redblood cells and the combined data obtained across the monkey studies,the primary treatment-related changes (i.e., anemia) are considered tobe related to the pharmacological action of Hu5F9-G4 binding to CD47expressed on RBCs. We believe that the administration of Hu5F9-G4accelerates the process of elimination of aging RBCs by substitutinggradual loss of CD47 with immediate blockade of CD47 on aging RBCs. Thepremature loss of aging RBCs is compensated by an ensuingreticulocytosis (which was observed across all studies), and over time,the initial anemia resolves as the aged RBCs are replaced with youngercells, and as a result, the age distribution of the RBC pool is shiftedto younger cells.

Despite the anemia related to administration of Hu5F9-G4, no evidence oftoxicity was observed in clinical signs in any animal, and thus,Hu5F9-G4 was clinically well-tolerated, even at doses as high as 300mg/kg. While administration of Hu5F9-G4 resulted in a transient anemia,no clinical signs of toxicity were noted, and Hu5F9-G4 was clinicallywell-tolerated by monkeys, even a doses as high as 300 mg/kg. Thetoxicology studies, however, did reveal that a small number of animalsare particularly sensitive to the Hu5F9-G4-associated anemia. While itis unknown at this time why these monkeys are more sensitive to theanemia produced by Hu5F9-G4, the anemia is transient, and consistentlyshowed a trend to recover upon termination of dosing. Since certainpatients may be more sensitive than others, changes in hematology willbe rigorously monitored in the proposed clinical study, and appropriatemeasures will be taken for any patient developing anemia belowpre-specified levels.

The anemia related to Hu5F9-G4 was partially or completely reversibleacross all of the monkey studies, and resolved for animals that wereplaced on dose holiday, including those animals where dosing hadresumed. The HNSTD for the 8-week toxicology study was thepriming/maintenance dose of 5/100 mg/kg (the highest dose tested). Basedon toxicokinetics data, the 5 mg/kg priming dose used in the 8-weektoxicology study is predicted to provide a safety margin (AUC-based)ranging from 28- to 194-fold above the proposed starting priming dose of0.1 mg/kg in the planned clinical trial. The twice weekly maintenancedose of 100 mg/kg provides a predicted safety margin (based on AUC)ranging from 766-803-fold above the starting maintenance dose of 0.1mg/kg planned for the proposed clinical study.

In summary, based on the results from the toxicology studies, thenonclinical safety assessment program supports the administration ofHu5F9-G4 (e.g., as an IV infusion) for the proposed clinical trial.

What is claimed is:
 1. A method of treating a mammalian subject withcancer with a therapeutic dose of anti-CD47 agent that blocks theinteraction between CD47 and SIRPα, selected from: an antibody thatbinds to CD47; an antibody that binds to SIRPα; a soluble SIRPα-bindingCD47 fragment; and a soluble CD47-binding SIRPα fragment or acombination thereof, for treatment of the cancer, the method comprising:(a) administering an erythropoiesis-stimulating agent (ESA) to thesubject in a dose effective to increase production of reticulocytes; and(b) administering a therapeutically effective dose of at least one ofthe anti-CD47 agents to the subject, wherein step (b) is performed in arange from about 3 days to about 21 days after beginning step (a). 2.The method according to claim 1, wherein step (b) is performed in arange from about 6 days to about 8 days after beginning step (a).
 3. Themethod according to claim 1, further comprising after step (a) and priorto step (b): a step of determining whether administration of the ESA waseffective by measuring in a blood sample from the subject one or moreof: an increase in the absolute or relative number of reticulocytes; anincrease in the level of EPO; and a decrease in hemoglobin levels. 4.The method according to claim 3, wherein the determining step comprisesperforming a reticulocyte count, wherein administration of the ESA isdetermined to have been effective if the reticulocyte count is about 400×10⁹ reticulocytes per liter (L) or more.
 5. The method according toclaim 3, wherein the determining step is performed in a range from about3 days to about 12 days after beginning step (a).
 6. The methodaccording to claim 1, further comprising after step (a) and prior tostep (b): administering a second dose of an erythropoiesis-stimulatingagent (ESA) to the subject effective to increase production ofreticulocytes to the subject wherein step (b) is performed in a rangefrom about 3 days to about 21 days after administering the secondsub-therapeutic dose.
 7. The method according to claim 1, wherein step(b) comprises administering the anti-CD47 agent in two or more doses ofescalating concentration until a therapeutically effective dose isadministered.
 8. The method according to claim 1, wherein step (b)comprises administering two or more therapeutically effective doses. 9.The method according to claim 1, comprising administering in thetherapeutically effective dose of (b) a combination of two or more ofthe anti-CD47 agents to the subject.
 10. The method of claim 1, whereinthe anti-CD47 agent specifically binds CD47.
 11. The method of claim 10,wherein the anti-CD47 agent is an antibody.
 12. The method of claim 10,wherein the anti-CD47 agent is a soluble CD47-binding SIRPα fragment.13. The method of claim 1, wherein the mammalian subject is a human. 14.The method of claim 1, wherein the ESA is erythropoietin.
 15. The methodof claim 1, wherein the ESA is administered at a dose from 50 units/kgto 17,000 units/kg of body weight.